Sphingobacterium multivorum - Journal of...

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JB00194-07 1

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Molecular Characterization of Membrane-Associated Soluble Serine Palmitoyltransferases 3

from Sphingobacterium multivorum and Bdellovibrio stolpii

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Hiroko Ikushiro*1, Mohammad Mainul Islam

1†, Hiromasa Tojo

2 and Hideyuki Hayashi*

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1Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan. Tel.: 7

+81-72-684-7291; FAX: +81-72-684-6516 8

2Department of Biochemistry and Molecular Biology, Osaka University, Graduate School of 9

Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan 10

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Running Title: Membrane-localized Bacterial Serine Palmitoyltransferases 12

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Footnotes 14

* Corresponding author. Mailing address: H. Ikushiro or H. Hayashi, Department of 15

Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan 16

TEL:+81-72-684-7291; FAX:+81-72-684-6516; E-mail: [email protected] or 17

[email protected] 18

†Present address: Department of Biochemistry, Wake Forest University School of Medicine, 19

Winston-Salem, NC 27157, USA 20

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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00194-07 JB Accepts, published online ahead of print on 8 June 2007

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Abstract: Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis 1

and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A (CoA) to 2

form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated 3

heterodimers belonging to the pyridoxal 5!-phosphate (PLP)-dependent !-oxamine synthase 4

family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant 5

water-soluble homodimeric SPT of the same family (Ikushiro et al., J. Biol. Chem. 276, 6

18249–18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, 7

although single crystals appropriate for high-resolution crystallography have not yet been 8

obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, 9

Sphingobacterium spiritivorum and Bdellovibrio stolpii, respectively. Each gene product 10

exhibits an ~30% sequence identity to both eukaryotic subunits, and the putative catalytic amino 11

acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia 12

coli and purified as water-soluble active homodimers. The spectroscopic properties of the 13

purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial 14

SPTs was confirmed by HPLC/mass spectrometry. The Sphingobacterium SPTs obeyed normal 15

steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate 16

inhibition at palmitoyl-CoA concentrations higher than 100 µM, as does the eukaryotic enzyme. 17

Immunoelectron microscopy showed that, unlike the cytosolic Sphingomonas SPT, S. 18

multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral 19

membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the 20

membrane-associated eukaryotic SPT. 21

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INTRODUCTION 1

Sphingolipids are ubiquitous membrane components of the eukaryotic plasma membrane (1), 2

and are known to be essential lipidic signaling molecules required for various cellular events 3

such as proliferation, differentiation, and apoptosis (2–4). In addition, sphingolipids together 4

with cholesterol are the major components of the membrane microdomains called "lipid rafts", 5

which serve as platforms for signal transduction or the transport of various bioactive molecules 6

via membrane trafficking (5–7). 7

Serine palmitoyltransferase (SPT: EC 2.3.1.50) catalyzes the pyridoxal 5!-phosphate 8

(PLP)-dependent condensation reaction of L-serine with palmitoyl coenzyme A (CoA) to 9

generate 3-ketodihydrosphingosine (KDS). This reaction is the first committed step in the de 10

novo biosynthetic pathway of all sphingolipids, producing long-chain bases (LCB), the backbone 11

structure of sphingolipids. SPT is thought to be the key enzyme regulating the cellular 12

sphingolipid content (8). Eukaryotic SPTs are enriched in the endoplasmic reticulum with their 13

catalytic sites facing the cytosol (9) and function as heterodimers comprising two tightly 14

membrane-bound subunits called LCB1 and LCB2, which share a sequence similarity (~25% 15

identity) (10–15). Recently, a new subunit protein of the human SPT, SPTLC3 has been found 16

(16). Due to the high sequence similarity (68 % identity) between SPTLC2 (LCB2 subunit of 17

human SPT) and SPTLC3, SPTLC3 is thought to form a dimer with SPTLC1. LCB2 (SPTLC2) 18

and SPTLC3 are the putative catalytic subunit carrying a lysine residue that forms the Schiff 19

base with PLP. In contrast, LCB1 does not have such a motif (10,14), and does not seem to 20

function as the catalytic center. Nevertheless, LCB1 is regarded to be essential for the catalytic 21

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action of SPT (15) and mutations in the LCB1 gene are known to cause human hereditary 1

sensory neuropathy type I (HSN1) (17–19). The roles of the SPT activity in the pathogenesis of 2

HSN1, however, are elusive at present (20–22). Elucidation of the structure-activity relationship 3

of SPT is essential for understanding the role of the rate-limiting enzyme, SPT, in regulating the 4

cellular sphingolipid homeostasis, as well as for clarifying the underlying causes of HSNI. There 5

is, however, little structural and mechanistic information on the mammalian SPT is currently 6

available, because the instability and the hydrophobic nature of each subunit have hindered the 7

successful purification of the recombinant SPT for crystallization and structural analysis (25). 8

Previously, we found and isolated a water-soluble homodimeric SPT from Sphingomonas 9

paucimobilis EY2395T (26). The Sphingomonas enzyme was successfully overproduced in E. 10

coli (26, 27). This bacterial prototype of the eukaryotic SPT provided a simple model system to 11

study the enzyme reaction without detergent micelles or lipid membranes. However, despite the 12

successful elucidation of the enzymological properties of the Sphingomonas SPT (28), we were 13

unable to obtain crystals appropriate for a high-resolution X-ray analysis, which is essential for 14

further clarification of the detailed catalytic mechanism of the enzyme. Therefore, we searched 15

for SPT proteins that are suitable for crystallization in other sphingolipid-containing bacteria. 16

One such candidate for the enzyme source is the genus Sphingobacterium, which is a deep 17

orange-pigmented rod belonging to the class of Sphingobacteria of the phylum Bacterioidetes 18

and is isolated from the environment (29) or from patients with opportunistic infections (30–34). 19

Sphingobacterium has a high concentration of sphingophospholipids with unique branched LCBs, 20

including ceramide phosphorylethanolamines, ceramide phosphoryl-myo-inositols, and ceramide 21

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phosphoryl-1-"-mannose as the major components (35–38). Another candidate, Bdellovibrio, is 1

a small curved rod belonging to the delta subclass of the phylum Proteobacteria; that can be 2

found in diverse environments such as marine and fresh waters, sewage, and soil (39). 3

Bdellovibrio is characterized by the unique predatory behavior by which they invade various 4

other larger Gram-negative bacteria, and grow as parasites in the intraperiplasmic space of the 5

prey (39–42). Bdellovibrio contains a phosphono ceramide, which carries the characteristic head 6

group of 1-hydroxy-2-aminoethyl phosphonate (43). The bacteria listed above are exceptions in 7

Gram-negative bacteria in that they lack lipopolysaccharides (LPSs) and instead contain a large 8

amount of sphingolipids including glycosphingolipids (GSLs) (36, 44–48); most Gram-negative 9

bacteria contain LPS, the major pathogenic glycolipids of the outer membrane. GSLs, such as 10

!-D-glucuronosyl-ceramide and !-D-galacturonosyl-ceramide of Sphingomonas, were reported 11

to activate CD1d-restricted natural killer T (NKT) cells (49–52). The action of the bacterial 12

sphingolipids in innate immunity is attracting much attention in the field of infectious diseases. 13

In this context, studying the SPT proteins in these bacteria is important for not only 14

obtaining the ideal source for crystallization of the enzyme, but also for providing the structural 15

basis for the elucidation of the biosynthetic mechanism of the unique glycosphingolipids in these 16

bacteria. We now report the molecular cloning of three novel SPTs from 17

sphingolipid-containing bacteria, Sphingobacterium spiritivorum EY3101T (S. spiritivorum), 18

Sphingobacterium multivorum GTC97 (S. multivorum) and Bdellovibrio stolpii ATCC 27052 (B. 19

stolpii). All of these bacterial enzymes were successfully overproduced in E. coli and 20

enzymatically characterized. Their properties resembled those of the eukaryotic enzyme more 21

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closely than the Sphingomonas enzyme. Thus, these enzymes can be useful models for 1

mammalian SPT and candidates for high-resolution crystallographic analyses. 2

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MATERIALS AND METHODS 5

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Chemicals. L-Serine and the other natural L-amino acids were obtained from Nacalai Tesque 7

(Kyoto, Japan). Palmitoyl CoA and lauroyl CoA were from Funakoshi (Tokyo, Japan). 8

Isopropyl 1-thio-"-D-galactoside (IPTG), myristoyl CoA, n-heptadecanoyl CoA, stearoyl CoA, 9

arachidoyl CoA, palmitoleoyl CoA, and oleoyl CoA were from Sigma. The LMW gel filtration 10

calibration kit, gel filtration calibration kit, PD-10, and Sephacryl S-200 HR were from 11

Amersham Bioscience/GE Healthcare. DEAE-Toyopearl 650M and butyl Toyopearl 650M were 12

from Tosoh (Tokyo, Japan). The competent E. coli JM109 was purchased from Nippon Gene 13

(Tokyo, Japan). E. coli BL21(DE3) pLysS and plasmids pET21b and pET28b were from 14

Novagen. Plasmid pUC118 was from Takara Bio (Kyoto, Japan). All other chemicals were of 15

the highest grade available from commercial sources. 16

Bacterial Strains and Growth Conditions. Sphingomonas paucimobilis EY2395T and S. 17

spiritivorum EY3101T were gifts from Dr. Eiko Yabuuchi (Gifu University School of Medicine, 18

Gifu, Japan). S. multivorum GTC97 was from the Gifu Type Culture Collection of Gifu 19

University, Japan. These three strains were aerobically grown in Bacto# heart infusion broth 20

(Difco, Becton Dickinson, MD) at 30 °C. B. stolpii (ATCC 27052) was purchased twice from 21

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ATCC, but it did not grow from the freeze-dried stock. A living culture was a gift from Dr. 1

Yoko Watanabe (Niigata University, Niigata, Japan). This strain was grown in culture medium 2

containing 1% Bacto# tryptone and 0.3% Bacto# yeast extract (Difco, Becton Dickinson, MD) 3

at 30 °C. The cells were collected by centrifugation and stored at –20 °C before use. 4

Isolation and Sequencing of Genomic DNA Clones Encoding S. multivorum SPT. The 5

genomic DNA of S. multivorum was prepared by a standard method (53). Based on the amino 6

acid sequences of the Sphingomonas SPT and eukaryotic LCB1/LCB2 proteins, we synthesized 7

degenerate oligonucleotides to obtain partial DNA fragments encoding the S. multivorum SPT 8

gene by PCR with genomic DNA from S. multivorum. The oligonucleotides 9

5!-GG(TCAG)(TA)(CG)(TCAG)TA(TC)AA(TC)TA(TC)(TA)T(AG)GG(TCAG)(TA)T-3! and 10

5!-CC(TCAG)AT(TCAG)G(TA)(AG)TG(TCAG)GC(TC)TC(AG)TC-3! corresponded to the 11

amino acid sequences GSYNYLMGF and DEAHSMG, respectively, of the Sphingomonas SPT. 12

PCR was performed using the LA Taq DNA polymerase (Takara Bio, Kyoto, Japan) under the 13

following conditions: 30 cycles of 94 °C for 30 s, 40 °C for 30 s, and 72 °C for 1 min, then 14

72 °C for 10 min. The PCR product was directly cloned into a pCRII vector (Invitrogen, The 15

Netherlands) and sequenced using a DYEnamic™ ET Dye Terminator Cycle Sequencing Kit 16

(Amersham Biosciences) and an ABI 310 DNA sequencer (Perkin-Elmer). To obtain the 17

full-length SPT gene, a genomic DNA library (3 $ 104 recombinants) was screened with the 18

32P-labeled PCR product (500 bp) as a probe. The library was constructed as follows: Genomic 19

DNA from S. multivorum was partially digested with Sau3AI, and fragments between 2.5 and 3.5 20

kb were purified by agarose gel electrophoresis and ligated into the BamHI-digested pUC118, 21

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and these constructs were used to transform the E. coli JM109. Labeling of the probe and 1

detection of the hybridizing fragments were performed using the BcaBEST™ labeling kit 2

(Takara Bio, Kyoto, Japan) and Quick-Hyb® hybridization solution (Stratagene), respectively. 3

Three positive clones were isolated in the first screening, and the complete DNA sequence was 4

determined for both strands of all three clones. 5

Isolation and Sequencing of Genomic DNA Clones Encoding the S. spiritivorum SPT. 6

Genomic DNA from S. spiritivorum was prepared by ISOPLANT (Wako, Osaka, Japan) 7

according to the manufacturer’s specifications. Based on the amino acid sequences of 8

Sphingomonas paucimobilis and S. multivorum SPTs, we synthesized degenerate 9

oligonucleotides to obtain partial DNA fragments encoding the S. spiritivorum SPT gene by PCR 10

with genomic DNA from S. spiritivorum. The oligonucleotides, 11

5!-CCA(TC)GC(TCAG)TC(AG)AT(TC)(TA)(TA)(TC)GA(TC)G-3! and 12

5!-CC(AG)CC(GT)A(TCAG)(TA)G(TA)(GT)(CG)C(TCAG)A(CA)CGA(TC)TT-3!, 13

corresponded to the amino acid sequences HASIID and KSLASLGG, respectively, of the S. 14

multivorum SPT. PCR was performed using the LA Taq DNA polymerase under the following 15

conditions: 30 cycles of 94 °C for 30 s, (40+ t) °C for 30 s, and 72 °C for 1 min, then 72 °C for 16

10 min, where t denotes that the annealing temperature was successively increased by 0.25 °C 17

for each cycle. The PCR product was cloned and sequenced. To obtain the full-length SPT gene, 18

a genomic DNA library (4 $ 104 recombinants) was screened with the

32P-labeled PCR product 19

(342 bp) as a probe. The construction of the genomic DNA library, labeling of the probe and 20

detection of the hybridizing fragments were carried out in the same way as for the S. multivorum. 21

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Two positive clones were isolated in the first screening, and the complete DNA sequence was 1

determined for both strands of both clones. 2

Isolation and Sequencing of Genomic DNA Clones Encoding the B. stolpii SPT. Genomic 3

DNA from B. stolpii was prepared by ISOPLANT according to the manufacturer's specifications. 4

Based on the amino acid sequences of the bacterial SPTs, we synthesized degenerate 5

oligonucleotides to obtain partial DNA fragments encoding the B. stolpii SPT gene by PCR with 6

genomic DNA from B. stolpii. The oligonucleotides, 7

5!-TGG(CA)TCACG(GT)(AT)T(CG)(TC)T(CA)AACGG(TC)AC(CG)TT-3! and 8

5!-CGAC(AG)AA(GT)CC(AG)CC(GT)A(CA)TG(AT)(GT)(CG)C(GT)A(CA)CGATTTTGA-3!, 9

corresponded to the amino acid sequences GSRFLNGTLD and SKSLASLGGFVA, respectively, 10

of the S. multivorum SPT. The PCR conditions were the same as those for the S. spiritivorum 11

SPT gene. To obtain the full-length SPT gene, a genomic DNA library (1 $ 105 recombinants) 12

was screened with the 32

P-labeled PCR product (536 bp) as a probe. Other methods to isolate the 13

SPT gene were the same as described above. Twenty-eight positive clones were isolated in the 14

first screening, and the complete DNA sequence was determined for both strands of the three 15

longest clones. 16

Expression of S. multivorum, S. spiritivorum and B. stolpii SPT Genes in E. coli. In order to 17

construct an expression system for the bacterial SPTs in E. coli, new restriction sites, NdeI and 18

EcoRI (for the S. multivorum SPT) or NdeI and SalI (for the S. spiritivorum and B. stolpii SPTs) 19

were respectively introduced into each SPT gene at the translation initiation and termination sites 20

by PCR. The internal NdeI restriction site (889

CATATG) of the S. spiritivorum SPT gene was 21

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changed to CACATG without changing the codons by site-directed mutagenesis using the 1

QuikChange® Site-Directed Mutagenesis Kit (Stratagene). Each modified DNA fragment was 2

ligated into the pET21b or pET28a vector using the LigaFast™ Rapid DNA Ligation System 3

(Promega), and each recombinant plasmid was used to transform the E. coli BL21 (DE3) pLysS 4

cells. The protein expression was induced with 0.1 mM IPTG and continued for 6 h at 37 °C. 5

Purification of Recombinant SPTs. All purification procedures were performed at 4 °C. All 6

buffers used for the purification contained 0.1 mM EDTA, 5 mM DTT, and 20 µM PLP unless 7

otherwise indicated. For the purification of the B. stolpii SPT, 20% (w/v) glycerol was added to 8

the purification buffers. The cells (10–25 g wet weight) were suspended in 150 ml of 20 mM 9

potassium phosphate buffer (pH 7.6) and disrupted by sonication (Branson Sonic Power, Sonifier 10

model 450) at 20 kHz for 3 min $ 3 times. The intact cells and debris were removed by 11

centrifugation (100,000 $ g, 60 min). The supernatant solution was applied to a 12

DEAE-Toyopearl 650M column (2.5 $ 20 cm) equilibrated with the same buffer. The proteins 13

were eluted with a linear gradient of 0 to 500 mM NaCl in 1 L of 20 mM potassium phosphate. 14

The fractions containing the SPT were collected. (NH4)2SO4 was added to 30% saturation, and 15

the solution was applied onto a Butyl-Toyopearl 650M column (2.5 $ 20 cm) equilibrated with 16

the same buffer containing 30%-saturated (NH4)2SO4. For the S. spritivorum SPT, the condition 17

of 20%-saturated (NH4)2SO4 was adapted. SPT was eluted with a decreasing linear gradient of 18

(NH4)2SO4 concentrations (30 to 0% or 20 to 0%) in 1 L of 20 mM potassium phosphate. The 19

pooled fractions were concentrated and then applied to a hydroxyapatite column (1.6 $ 20 cm) 20

equilibrated with 10 mM potassium phosphate buffer (pH 7.6). The proteins were eluted with a 21

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linear gradient of 10 to 250 mM potassium phosphate in 1 L. The SPT fractions were 1

concentrated and then applied to a Sephacryl S-200 HR column (1.6 $ 80 cm) equilibrated with 2

50 mM potassium phosphate buffer (pH 7.6) containing 0.1 mM EDTA and 150 mM NaCl. The 3

active fractions were combined, concentrated to 2–5 ml, filtered, and stored at 4 °C. 4

Mass Spectrometric Analyses of Reaction Products. To identify the reaction products of the 5

bacterial SPT, the reaction was carried out in the presence of 1.6 mg purified SPT, 20 mM 6

L-serine, 5 mM acyl CoA, 50 mM EDTA, 50 mM HEPES, 0.15 M KCl (pH 7.5) in a final 7

volume of 0.1 ml. The reaction was stopped by the addition of 0.1 ml of ~2 M ammonia, and 8

then the total lipids were extracted by the method of Bligh and Dyer (54) followed by 9

hexane/2-propanol (3:2 v/v) and a salt solution partition (55). The lipid products were identified 10

by electrospray ionization (ESI)/ ion-trap mass spectrometry connected online to the 11

normal-phase HPLC (56, 57). LCBs can be analyzed by a method similar to the ceramide 12

analysis previously reported (56) with minor modifications. A trap column (1 x 20 mm) fitted to 13

a switching valve (Valco Instruments Co., Houston, Texas, USA) was preequilibrated with 14

solvent A (hexane containing 0.1% formic acid). An aliquot of the extracted lipids was applied 15

onto the trap and then washed with the same solvent. Immediately after connecting the trap by 16

valve switching to a separation silica column (1 " 150 mm, OmniSeparo-TJ, Hyogo, JAPAN) 17

equilibrated with solvent A, the LCBs bound to the trap were eluted with the following gradient 18

sequence: from 100% solvent A to 70% solvent A and 30% solvent B (hexane:2-propanol; 4:6 19

by volume) in 6 min, to 95% solvent B and 5% solvent C (hexane:2-propanol:1 M ammonium 20

formate:water; 40:60:2.24:9.76 by volume) in 5 min, then to 50% solvent B and 50% solvent C 21

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in 15 min. The effluent was monitored by a ThermoElectron LCQdeca mass spectrometer 1

equipped with an ESI tip, FortisTip (20 mm ID and 150 mm OD, OmniSeparo-TJ, Hyogo, 2

JAPAN) on a xyz stage (AMR, Tokyo, JAPAN) in positive-ion full scan and data-dependent 3

positive-ion MS/MS modes on a single run. An ESI voltage of 1.6 kV was used. 4

Spectrophotometric Measurements. The absorption spectra of the SPTs were recorded by a 5

Hitachi U-3300 spectrophotometer at 25 °C. The circular dichroism (CD) spectra of the SPTs 6

were recorded by a Jasco J720-WI spectropolarimeter at 25 °C. The buffer solution for the 7

spectrometric measurements contained 50 mM HEPES-NaOH (pH 7.5), 150 mM KCl, and 0.1 8

mM EDTA. The purified enzyme was equilibrated with this buffer by gel filtration using a 9

PD-10 (Sephadex G-25) column prior to the measurements. 10

Antibody Preparation against each Bacterial SPT. The antiserum against Sphingomonas 11

paucimobilis, S. multivorum, or B. stolpii SPT was prepared by immunization of rabbits with the 12

purified SPT proteins. Each anti-SPT IgG was affinity-purified with the corresponding 13

SPT-immobilized Sepharose 4B from the total IgG fraction of the rabbit serum by a standard 14

method. 15

Immunoblot Analysis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as 16

described by Laemmli (58) with the SDS-Tris system using a 3% (w/v) stacking and a 10% (w/v) 17

separating gel. The gels were blotted onto Immobilon-PSQ

PVDF membranes (Millipore, MA, 18

USA) using a semi-dry blotting method. The membranes were blocked at room temperature for 19

2 h in PBS containing 1.5% (w/v) BSA, followed by incubation with diluted anti-SPT rabbit 20

antibodies at room temperature for 3 h. The membranes were washed and incubated with a 21

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1:5000 dilution of the horseradish peroxidase-conjugated goat anti-rabbit antibody solution at 1

room temperature for 3 h. Visualization of the immunoreactive bands was performed using 2

chemical luminescence (ECL detection kit, Amersham Biosciences Inc., Piscataway, NJ). 3

Electron Microscopic Analysis by the Thin-section (Postembedding) Method. The cells were 4

first fixed in a 0.1 M sodium cacodylate buffer (pH 7.4) containing 4% paraformaldehyde and 5

0.1% glutaraldehyde for 2 h at 4 °C, dehydrated in a graded series of ethanol solutions (50% to 6

100%), and embedded in LR White resin (London Resin Co., Ltd., Hampshire, UK) overnight at 7

60 °C. Ultrathin sections (90 nm in thickness) were prepared by an ultramicrotome (2088-V, 8

LKB, Bromma, Sweden). The sections were transferred onto fine nickel grids, incubated in PBS 9

containing 1% BSA and 1.5% normal goat serum for 15 min at room temperature and in PBS 10

containing 1% BSA, 1.5% normal goat serum and anti-SPT IgG overnight at 4 °C. Subsequently, 11

the grid was incubated in PBS containing 1% BSA, 1.5% normal goat serum and goat anti-rabbit 12

IgG conjugated to 10-nm gold particles for 30 min at room temperature. All sections were 13

finally stained with uranyl acetate and lead citrate. The preparation was examined using an 14

electron microscope, JEM2000EX (JEOL, Tokyo, Japan). 15

Other Methods. The SPT activity was measured according to previously described methods (26). 16

The protein concentration during the purification procedure was determined using a BCA protein 17

assay kit (Pierce Chemical) with bovine serum albumin as the standard. The protein 18

concentration of the purified SPT was spectrophotometrically determined using the following 19

molar extinction coefficients at 280 nm for the PLP form of each enzyme: 2.83 $ 104 M

–1% cm–1

20

for S. paucimobilis SPT (26); 2.68 $ 104 M

–1% cm–1

for S. multivorum SPT; 2.68 $ 104 M

–1% cm–1

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for S. spiritivorum SPT; 2.68 $ 104 M

–1% cm–1

for B. stolpii SPT. 1

2

RESULTS 3

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SPT activity in S. multivorum and B. stolpii. We previously reported that both Sphingomonas 5

paucimobilis SPT and S. spiritivorum SPT are water-soluble enzymes (26). The SPT activities in 6

S. multivorum and B. stolpii were examined in a similar way, and the results are presented in Fig. 7

1 together with those of Sphingomonas paucimobilis and S. spiritivorum, and the mouse liver 8

microsome (reference). For all the bacterial strains, the SPT activity was found in both the 9

supernatant and precipitate fractions, which were prepared by ultracentrifugation. The 10

distribution profile of the activity in the supernatant and the precipitate seems to vary depending 11

on the species. However, as the precipitate fraction contains a non-negligible amount of unlysed 12

cells, it is hard to precisely estimate how much of the SPT activity is associated with the 13

membrane. This issue was morphologically assessed using immunoelectron microscopy, as 14

described later in detail. 15

Cloning of SPT Genes from S. multivorum, S. spiritivorum and B. stolpii. The three SPT 16

genes (spt) from S. multivorum, S. spiritivorum and B. stolpii were cloned by degenerate-PCR 17

and genome library screening, as described in the Materials and Methods section. The 18

nucleotide and protein sequences of the spt genes have been submitted to the GenBank database 19

(S. multivorum, AB259214; S. spiritivorum, AB259215; B. stolpii, AB259216). Properties of 20

the gene products of the bacterial SPTs are summarized in Table 1. 21

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Sequence Comparisons. The amino acid sequence alignment of the human SPT subunits, 1

SPTLC1/SPTLC2/SPTLC3, isolated bacterial SPT genes and the putative SPT gene of 2

Zymomonas mobilis ZM4 (59), and two other enzymes of the !-oxamine synthase family, i.e, E. 3

coli 8-amino-7-oxonanoate synthase (AONS) and human 5-aminolevulinate synthase (ALAS), is 4

shown in Fig. 2A. AONS and ALAS were selected as the proteins most and second most 5

homologous to SPT, respectively. An overall sequence similarity was found between these 6

proteins. Bacterial SPTs are highly similar to each other, and conserved amino acids are 7

distributed throughout the entire sequences of the polypeptides. The S. multivorum SPT shared 8

the highest amino acid sequence identity (87.7%) with the S. spiritivorum SPT. The Z. mobilis 9

SPT also showed a high identity (73.1%) with the Sphingomonas SPT. The amino acid sequence 10

identities among other bacterial SPTs were 35.0–48.2%. The conservation of the amino acid 11

sequences between each bacterial enzyme was higher than that between the bacterial SPTs and 12

human SPTLC proteins. S. multivorum SPT is 25%, 31%, and 32% identical, S. spiritivorum 13

SPT is 27%, 32%, and 33% identical, and B. stolpii SPT is 24%, 33%, and 34% identical to the 14

human SPTLC1, SPTLC2, and SPTLC3, respectively. Several small hydrophobic stretches of 15

amino acids were distributed throughout the bacterial SPT protein; however, no obvious 16

transmembrane region(s) were found. The SPT-specific PLP-binding motif 17

(GTFSKSXXXXGG) is completely conserved among all the bacterial SPTs. 18

A phylogenetic tree of the SPTs was constructed by the neighbor-joining method using the 19

E. coli AONS protein as an outgroup (Fig. 2B). The selection of this protein as the outgroup is 20

because E. coli AONS is the protein apparently distinct from SPT, but has the highest similarity 21

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to SPT among the !-oxamine synthase family enzymes. The bootstrap values at the nodes 1

except for the leftmost two nodes were all 100%. The reason for the relatively low values of 2

74% and 82% of the two nodes is not clear, but may be partially attributed to the use of the 3

evolutionarily distant (i.e., functionally different) outgroup. The divergence of the bacterial 4

SPTs reflects the phylogeny of the bacteria; Sphingomonas paucimobilis and Z. mobilis belong to 5

the same family of bacteria called Sphingomonadaceae, and S. multivorum and S. spiritivorum 6

belong to the family Sphingobacteriaceae (48, 59). The branch lengths of the LCB1 proteins are 7

significantly longer than those of other proteins including the LCB2, SPTLC3, and bacterial 8

SPTs, suggesting relatively higher evolution rates of the LCB1 proteins. S. spiritivorum SPT is 9

the nearest relative to the mammalian LCB2 proteins, followed by the SPTs from S. multivorum, 10

B. stolpii, Z. mobilis and Sphingomonas paucimobilis. 11

Overproduction and Purification of Recombinant SPTs. Each bacterial SPT has been stably 12

overexpressed as a soluble protein in E. coli. The expression levels of the recombinant proteins 13

reached approximately 10–20% of the total protein of E. coli cells without growth inhibition of 14

the expression host. While the B. stolpii SPT was most abundantly expressed, half of the 15

expressed protein formed inclusion bodies. In order to increase the solubility of the recombinant 16

enzyme, we made a deletion variant of the B. stolpii SPT lacking the N-terminal 13 amino acid 17

residues. The addition of 20% (w/v) glycerol was necessary to prevent the precipitation of the B. 18

stolpii SPT during purification and storage. All the recombinant enzymes were purified to 19

homogeneity by column chromatography in three steps, and the purified SPTs showed a single 20

protein band with an apparent Mr of approximately 45,000 for the S. multivorum and S. 21

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spiritivorum SPTs and 50,000 for the B. stolpii SPT, respectively, on SDS-PAGE (data not 1

shown). About 20 mg of purified enzyme were routinely obtained from 1-L cultures in each case, 2

and could be stored at 4 °C for more than 6 months. 3

Physicochemical Characterizations. The Mr values of all three bacterial SPTs were estimated to 4

be 90,000 by gel filtration. MALDI-TOF MS analyses gave a signal at m/z 43,645 for the S. 5

multivorum SPT, 43,780 for the S. spiritivorum SPT, and 44,397 for the B. stolpii SPT lacking 6

the N-terminal 13 amino acid residues. These values were in good agreement with the values of 7

43,640, 43,797 and 44,522, which were calculated from the deduced amino acid sequences of 8

each recombinant enzyme without the first methionine within experimental error. These results 9

show that these bacterial SPTs have dimeric structures composed of two identical subunits. 10

The pH stability of the purified SPTs was investigated (data not shown). This information is 11

necessary both for crystallization and storage. Enzymes from both S. spiritivorum and B. stolpii 12

SPTs showed an above 90% activity in the pH range of 6.8–8.5, with an optimum pH at 7.0–8.0. 13

Only the S. multivorum SPT was denatured below pH 7.2, and its optimum pH was 7.4–8.0. 14

All purified recombinant SPTs showed characteristic absorption spectra of PLP-dependent 15

enzymes (Fig. 3). For all the bacterial SPTs, the intensities of the absorption peaks were not 16

changed by the pH. The shapes of the spectra of the two Sphingobacterium SPTs were different 17

from that of the Sphingomonas enzyme (Fig. 3A, B and C, solid line). They had only a single 18

peak at 426 nm in addition to the protein absorption peak at 278 nm. On the other hand, the B. 19

stolpii SPT showed two peaks at 338 and 426 nm (Fig. 3D, solid line), and in this respect, similar 20

to the Sphingomonas enzyme, although the relative intensities of the two peaks are different. 21

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These absorption peaks respectively correspond to the enolimine and the ketoenamine forms of 1

the internal Schiff base (aldimine formed between the aldehyde group of PLP and the &-amino 2

group of a lysine residue in the active site) of SPT. The addition of L-serine to the purified SPTs 3

gave rise to an intense absorption band at 426 nm for all the SPTs, and a weak band at 338 nm 4

for the Sphingomonas and B. stolpii SPTs (dashed lines). These spectral changes showed 5

hyperbolic dependencies on the concentrations of L-serine, and the apparent dissociation 6

constants (Kd) for L-serine were calculated to be 0.47, 1.20 and 2.55 mM, respectively, for the S. 7

multivorum, S. spiritivorum, and B. stolpii SPTs (Table 2). The CD spectra of these bacterial 8

SPTs showed positive bands at 426 nm (and additionally at 338 nm for B. stolpii SPT), 9

corresponding to the absorption spectra of each enzyme (data not shown). The CD spectra in the 10

presence of a saturating amount of L-serine showed a negative band at 426 nm (data not shown). 11

These results indicate that the added L-serine formed the external Schiff base (aldimine formed 12

between PLP and extraneously added amino acid) with PLP. The addition of the second substrate, 13

palmitoyl CoA, to the B. stolpii SPT, which is saturated with L-serine, resulted in a slight 14

decrease in the 426-nm peak and the appearance of an absorption band at 515 nm (Fig. 3C, 15

dash-dotted line). The formation of the 515-nm-peak was transient and vanished within a few 16

minutes. Such spectral changes were not observed for the Sphingobacterium SPTs or the 17

Sphingomonas SPT. 18

Identification of the Reaction Products of Bacterial SPTs. The formation of KDS by bacterial 19

enzymes was confirmed by HPLC/ESI–ion-trap mass spectrometry (Fig. 4). Fig. 4A shows the 20

ion chromatograms (m/z 300) of the SPT reaction product in the positive-ion mode. The most 21

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abundant ions at m/z 300 (Fig. 4C) corresponded to the protonated molecular ions [M + H]

+ of 1

C18:0 KDS formed from L-serine and palmitoyl-CoA, the structure of which is shown in Fig. 4B. 2

This LCB structure was confirmed by on-line MS/MS.

A positive-ion MS/MS spectrum of the 3

m/z–300 ion showed the presence of ions of m/z 282 and 270, arising from the neutral

loss of 4

H2O and HCHO, respectively (Fig. 4B and 4D). 5

Catalytic Properties of Bacterial SPTs. Like eukaryotic enzymes, all the bacterial SPTs used 6

only the L configuration of serine as an amino acid substrate. The bacterial enzymes exhibited a 7

broad substrate specificity concerning the chain length and the degree of unsaturation of acyl 8

CoA (Fig. 5). Fig. 5A shows ion chromatograms of the reaction product formed by the S. 9

multivorum SPT. In each chromatogram, a single ion peak of the reaction product with a 10

molecular weight corresponding to the value expected from the chain length of the acyl CoAs 11

was detected. The longer chain length of KDS gave a slightly shorter retention time. Fig. 5B 12

shows the MS data in which the molecular ion at m/z 356 corresponded to [M + H]+ of C22:0 13

KDS formed from L-serine and arachidoyl CoA. As shown in Fig. 5C, the MS/MS spectrum of 14

the m/z–356 ion indicated the presence of ions of m/z 338, 326 and 320, arising from the neutral

15

loss of H2O, HCHO and 2H2O, respectively. The structures of the other KDS with different chain 16

lengths were also confirmed in the same way. 17

Fig. 6 shows the substrate concentration dependency of the reaction rate of the bacterial 18

SPTs. It has been reported that eukaryotic SPT is inhibited by palmitoyl CoA concentrations 19

greater than 50 µM (15, 60). A similar phenomenon was observed with the B. stolpii SPT, which 20

was significantly inhibited by palmitoyl CoA concentrations greater than 100 µM (Fig. 6C). The 21

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SPTs of S. multivorum and S. spiritivorum were not inhibited by the high concentrations of 1

palmitoyl CoA. For the latter two enzymes, we could analyze the experimental data under 2

steady state conditions according to the ordered Bi-Bi mechanism (Fig. 6A, B) (61), and the 3

kinetic parameters were obtained (Table 2). For all the enzymes, the Km values for L-serine were 4

in the range of 3–5 mM. The kcat values of the S. multivorum and S. spiritivorum enzymes are 5

apparently lower than that of the S. paucimobilis enzyme. The Km values for palmitoyl CoA 6

were 0.1–0.7 mM, except for the B. stolpii enzyme. For the B. stolpii enzyme, the Km for 7

palmitoyl CoA and kcat could not be obtained due to the substrate inhibition by palmitoyl CoA. 8

For comparison with other data, the v/[Et] value in the presence of 100 µM palmitoyl CoA, 9

where the maximum activity is obtained, is shown. 10

Intracellular Localization of Bacterial SPTs (Immunolocalization). In order to examine the 11

localization of the SPTs in the intact cell, specific polyclonal antibodies were prepared using the 12

recombinant enzymes. As shown in Fig. 7, each antibody specifically recognized its 13

corresponding SPT. There was no signal in the lanes containing the E. coli lysate transformed 14

with the empty vector (Fig. 7, lanes 1, 4, and 7). The anti-S. multivorum SPT antibody 15

cross-reacted with the S. spiritivorum SPT (data not shown). Considering the phylogenetic 16

similarity of S. spiritivorum to S. multivorum that might yield similar morphological results, and 17

the technical constraints on the morphological analysis of this weak pathogenic species, S. 18

spiritivorum was excluded from further analyses. For the B. stolpii lysate, the antibody 19

recognized two bands of approximately 50 kDa and 48 kDa, the latter being the same size as the 20

recombinant SPT lacking the N-terminal 13 amino acid residues (Fig. 7, lanes 8 and 9). The thin 21

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sectioned profiles of the whole bacterial cells of Sphingomonas paucimobilis, S. multivorum and 1

B. stolpii are presented in Fig. 8A, B and C, respectively. Sphingomonas paucimobilis cells have 2

a rod shape, and the average size (width) of the cells was 0.8 µm (Fig. 8A). The cytoplasm of 3

the Sphingomonas paucimobilis cells was characterized by the presence of clearly identifiable 4

ribosome particles and a non-nucleoid electron-dense area. The cell envelope consisted of a one 5

electron-dense bilayer as an outer membrane and one additional bilayer structure as an inner 6

membrane. The ribosome particles showed a condensed distribution near the inner membrane or 7

the non-nucleoid electron-dense area. The shape of the S. multivorum cells was also rod-type, 8

and the average size (width) was 0.45 µm (Fig. 8B). The S. multivorum cells were slightly 9

shorter than the Sphingomonas paucimobilis cells and had no flagella. At the inside of the cell 10

wall, a multilayered inner membrane structure was observed. The B. stolpii cells had the shape 11

of curved rods or spheres with a rugged cell wall (Fig. 8C). The average size (width) of the cells 12

was 0.36 µm. Like Sphingomonas paucimobilis and S. multivorum, a multilayered cell 13

membrane structure was seen inside of the cell wall of the B. stolpii cells. The ribosome 14

particles were widely distributed in the electron-dense cytoplasm. In the middle of the 15

cytoplasm, an organelle-like multilayered membrane structure was observed. The intracellular 16

localization of SPT was analyzed for each bacterium using immunoelectron microscopy. The 17

immunogold-labeled SPT was readily detectable as a spot-like distribution throughout the 18

cytoplasm in the Sphingomonas paucimobilis cells (Fig. 8D). The non-nucleoid electron-dense 19

area observed in these bacterial cells was more intensely immunostained. Some of the 20

immunogold clusters seemed to localize near the inner membrane of the cell. Sparse 21

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immunogold particles were also detected on the outside of the cell. In the S. multivorum cells, 1

about 88% of the immunogold-labeled SPT was distributed near the inner membrane of the cell, 2

and the remaining immunogold particles were detected in the center of the cells (cytoplasm) (Fig. 3

8E). In the B. stolpii cells, the immunogold-labeled SPT was detected in a spot-like pattern 4

predominantly concentrated in a limited region near the inner membrane or organelle-like 5

multilayered membrane structure (Fig. 8F). When each primary antibody had been preabsorbed 6

with the corresponding SPT protein, the signals disappeared or at least became very faint (Fig. 7

8G–I). 8

9

DISCUSSION 10

11

Our recent efforts to crystallize SPT using the recombinant enzyme from Sphingomonas 12

paucimobilis, which we had previously characterized, showed that the stability of the 13

Sphingomonas SPT was not sufficient for crystallization and structural analysis. To obtain a 14

suitable protein for crystallization, we searched for SPTs from other bacterial sources. S. 15

multivorum, S. spiritivorum and B. stolpii have been reported to contain large amounts of 16

sphingolipids as cell membrane components (39, 43). Their SPT activities in the cytosolic 17

fractions were comparable to that of Sphingomonas paucimobilis. Based on the amino acid 18

sequences of the conserved regions between Sphingomonas SPT and the eukaryotic LCB1/LCB2 19

proteins, we carried out degenerate PCR and genomic library screening. Three novel SPT genes 20

were isolated from these bacteria. Each recombinant protein catalyzed the KDS formation from 21

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L-serine and palmitoyl CoA (Fig. 4), confirming that the products of all of the cloned genes have 1

SPT activity. 2

Catalytically Important Amino Acid Residues are Conserved in Bacterial SPTs. SPT belongs 3

to the !-oxamine synthase family of the PLP-dependent enzymes, which includes the 4

5-aminolevulinic acid synthase (ALAS), 2-amino-3-ketobutyrate ligase (KBL), and 5

8-amino-7-oxononanoate synthase (AONS) (62–67). Bacterial SPTs show about a 30% identity 6

with other members of this family. Previous X-ray crystallography on AONS and KBL from E. 7

coli suggested catalytically important active-site residues that interact with PLP and are 8

completely conserved in all the bacterial SPTs. These conserved residues include (in S. 9

multivorum numbering) Lys244, which forms a Schiff base linkage with PLP, Asp210, which 10

forms a salt bridge/hydrogen bond with the pyridine N of PLP, His213, which hydrogen bonds to 11

O3! of PLP, and His138, which stacks with the pyridine ring of PLP (Fig. 2A, reversed triangles). 12

The structures of the complexes of AONS and KBL with substrate analogues suggested that 13

Asn52 and Arg367 are the potential hydrogen-bonding partners of the carboxylate group of the 14

substrate L-serine in the external aldimine complex of SPT. Arg367 is indeed conserved in all 15

the SPTs, but Asn52 is partially conserved; it shifts by one residue in the Sphingomonas and Z. 16

mobilis SPTs. 17

The sequence similarities between the bacterial SPTs and human LCB2 are higher than 18

those between the bacterial enzymes and human LCB1. The active site residues described above 19

are not conserved in the human LCB1; Lys244, His213, and His138 are replaced by Asn, Leu, 20

and Cys, respectively. These residues in LCB1 cannot functionally substitute for the 21

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corresponding residues of the other !-oxamine family enzymes including LCB2. Apparently, 1

LCB1 does not have a catalytic function. This is consistent with the longer branch length of 2

LCB1, because the lack of functional constraints on the protein is considered to accelerate the 3

evolution rate. 4

Human Hereditary Sensory Neuropathy Type I-related Mutation Site of Bacterial SPT. The 5

single mutations (C133Y, C133W, V144D or G387A) in the human LCB1 cause HSN1, which is 6

the most common hereditary disorder of peripheral sensory neurons (17, 18, 19). It remains 7

elusive how the LCB1 mutations cause changes in the SPT activity of the heterodimer, and how 8

these changes participate in the neurodegenerative symptoms in HSN1. The dominant negative 9

inhibition of the SPT activities by overexpression of the HSN1-related LCB1 mutants in yeast, 10

CHO cells and transgenic mouse strongly supports the idea that the neurodegeneration in HSN1 11

is related to a decrease in the sphingolipid synthesis (20, 21, 22). However, there is a claim that 12

the remaining SPT activity is sufficient for the normal sphingolipid metabolism and viability of 13

the HSN1 patient cells (23). An alternative mechanism is that the HSN1 mutations of LCB1 14

accelerate the aggregation of the SPT protein induced by hypoxia in the human lymphocytes, 15

which leads to non-apoptotic death (24). Contrary to these observations, de novo glucosyl 16

ceramide synthesis increased in the lymphoblast cell lines from HSN1 patients (18), suggesting 17

that the neural degeneration in HSN1 is due to the overproduced ceramide or the abnormal 18

cellular lipid composition. Cys133 and Val144 of the human LCB1 correspond to Cys78 and 19

Ile90, respectively, of the S. multivorum SPT. These residues are also conserved in the SPTs of S. 20

spiritivorum and B. stolpii, but not in Sphingomonas paucimobilis and Z. mobilis sequences, in 21

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which Cys133 and Val144 correspond to Thr and Asp/Gly, respectively. It should be noted that 1

the Sphingomonas SPT, which carries an amino acid substitution equivalent to the HSN1-type 2

mutation (Asp at the position of Val144 in human Lcb1p), has a much higher activity than other 3

bacterial enzymes carrying a hydrophobic amino acid (Ile) at that position. These findings lead 4

us to speculate on the possibility that the HSN1-type mutations in the human LCB1 may result in 5

some toxic gain of function, such as increased activity toward normal or abnormal acyl-CoAs. 6

Immunolocalization of Bacterial SPT as a Peripheral Membrane Protein. All the bacterial 7

SPTs examined so far are water-soluble homodimeric enzymes. Their water-soluble character is 8

the most different aspect from the membrane-bound enzymes of the eukaryotes. The reaction 9

product of SPT, KDS, is a very hydrophobic sphingolipid intermediate that is easily incorporated 10

into membranes. While membrane localization of the eukaryotic SPT complex seems very 11

reasonable, the characteristics of the bacterial SPT as a water-soluble protein in vitro raised 12

questions about the mechanism of the product release or the interaction with the cellular 13

membrane in vivo. Therefore, we further analyzed the intracellular localization of the bacterial 14

SPT by immunoelectron microscopy (Fig.8). These results demonstrated the limited distribution 15

of SPT molecules in bacterial cells in a spot-like pattern (Sphingomonas paucimobilis), or a 16

clearly condensed pattern near the inner membrane of the cells (S. multivorum and B. stolpii). 17

These results are different from the homogenous distribution pattern of the general soluble 18

proteins. S. multivorum and B. stolpii SPT may loosely bind to the inner membrane of bacterial 19

cells like a peripheral membrane protein, or may indirectly interact with the membrane via some 20

anchor protein in vivo. The KDS released from these SPTs may directly enter the inner 21

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membrane, and then be efficiently converted into various glycosphingolipids as the final 1

products by other modification enzymes, which may present in the bacterial cell membrane. 2

The Most Eukaryotic-like SPT from B. stolpii. The SPT from B. stolpii is different from the 3

other bacterial SPTs in the following ways. First, during purification, the recombinant enzyme 4

required the addition of 20% glycerol, which is the most general stabilizer for membrane 5

proteins. Second, B. stolpii has an organelle-like multilayered membrane structure within the 6

cell, and the immunoreaction to the native SPT was detected on the cytosolic face of the 7

organelle-like structure. Third, the reaction rate is the lowest among the bacterial SPTs 8

examined, and is on the same order as the eukaryotic enzymes. Fourth, when both L-serine and 9

palmitoyl CoA were added to the enzyme, a transient accumulation of the quinonoid 10

intermediate was spectroscopically detected, suggesting a slow catalytic turnover of the enzyme. 11

Finally, the inhibition by palmitoyl CoA as seen in the mammalian SPTs was observed. These 12

data indicate that the B. stolpii SPT is the most eukaryotic-like enzyme among the bacterial STPs, 13

although it is a bacterial homodimeric SPT, and in this respect, different from the heterodimeric 14

enzymes of the eukaryotes. 15

The physiological function of the sphingolipids in bacteria is unknown except for their role 16

as a main component of the bacterial cell membrane. The broad acyl CoA specificity of bacterial 17

SPT might be advantageous to bacteria in that they can escape from the influence of the 18

environmental changes on bacterial envelope properties that affect their survival. If the fatty 19

acids available to the bacterial cells are changed, their SPTs can utilize other acyl CoAs with 20

different chain lengths for the LCB synthesis. On the other hand, there is no current report that 21

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these organisms produce sphingolipids of varying LCB lengths depending on the substrate fatty 1

acids to which they are exposed. Considering that the [14

C]-labeled palmitic acid in the culture 2

media is selectively incorporated into the LCB of Sphingomonas under normal conditions (46), it 3

seems more probable that these organisms make essentially the same sphingolipid LCBs 4

regardless of the fatty acid composition in the environment. This is a future research topic. 5

There is an interesting report demonstrating that the predatory bacterium, the Bdellovibrio 6

bacteriovorus UKi2 strain, containing phosphosphingolipids and the mutant UKi1 strain lacking 7

sphingolipids loses its parasitic ability (42). The sphingolipids of Bdellovibrio may be associated 8

in some way with the ability of this bacterium to attack and grow on suitable bacterial hosts. It 9

has been reported that the glycosphingolipids from Sphingomonas sp. and Ehrlichia muris were 10

recognized by the CD1d-restricted NKT cells in the mouse and human, which provide an 11

innate-type immune response (49–52). We have found putative SPT genes among the genome 12

databases of some pathogenic bacteria, and have already examined the SPT activity of these gene 13

products. These pathogenic bacteria may also have cell walls containing glycosphingolipids that 14

serve as direct targets for the NKT cells. Another example of the possible involvement of 15

sphingolipids in pathogenesis is also shown by a recent finding that the marine planktonic 16

pathogen, Coccolithovirus EhV-86, has a cluster of genes highly homologous to the enzymes of 17

the sphingolipid metabolism (70), and these genes are coordinately expressed within 2 h of 18

infection (71). This viral SPT is a unique monomeric enzyme, which is composed of two 19

separable domains, suggesting a fused heterodimer corresponding to the eukaryotic SPTs (72). 20

This viral SPT expressed in the yeast cell was localized to the endoplasmic reticulum, and 21

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preferred myristoyl CoA (C14) to palmitoyl CoA (C16) as the substrate. It was suggested that the 1

viral SPT may alter the sphingolipid metabolism of the host during pathogen infection. 2

3

We obtained novel SPT molecules with various characteristics, from the highly stable and 4

water-soluble type to the eukaryotic-like and loosely membrane-bound type. Recently, the 5

recombinant SPT from S. multivorum has yielded crystals of sufficiently good quality for X-ray 6

crystallographic analysis. Further structural analyses of the SPT-complex with the substrate, 7

product, or analogues are now under way. Not only the S. multivorum SPT, but also other 8

bacterial enzymes will be useful because they have the characteristic spectroscopic features 9

reflecting the intermediate accumulation of each step in the catalytic cycle. The reaction 10

mechanism of SPT could be clarified in the context of the three-dimensional structure of the 11

bacterial SPT. Information obtained from the bacterial enzymes will provide clues to the 12

reaction mechanism of the more complex eukaryotic homologue. 13

14

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16

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1

2

Acknowledgments 3

We thank Dr. Eiko Yabuichi of the Aichi Medical University and Prof. Yoshiaki Kawamura of 4

the Aichi Gakuin University for generously providing bacterial strains. We also acknowledge 5

Prof. Nobuyoshi Esaki and Dr. Tatsuo Kurihara of Kyoto University for the useful comment on 6

the immunoelecromicroscopic study. 7

8

9

This work was supported by a Grant-in-Aid for Encouragement of Young Scientists (B) 10

16770103 and a Grant-in-Aid for Scientific Research (C) 18570114 (to H. I.) from the Ministry 11

of Education, Culture, Sports, Science and Technology of Japan, and by a Grant-in-Aid for 12

Scientific Research (C) 16570125 (to H.H.) from the Ministry of Education, Culture, Sports, 13

Science, and Technology of Japan. 14

15

16

FIGURE LEGENDS 17

18

FIG. 1. Thin-layer chromatography of radiolabeled products obtained by the SPT assay 19

reactions of mouse liver microsomes, S. paucimobilis, S. multivorum, S. spiritivorum and B. 20

stolpii. The assay reactions and thin-layer chromatography were carried out as described in ref. 21

26. Reaction products formed in the presence of the crude extract, the precipitate (resuspended), 22

or the supernatant, were spotted. The volume of the crude extract used for the reaction was 100 23

µl (140 mg protein/ml), and the amounts of the precipitate and the supernatant were those 24

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obtained from the same volume (100 µl) of the crude extract. Lanes 1 and 8, mouse liver 1

microsomes as a reference; lanes 2, 5, 9, and 12, crude extracts after sonication of S. 2

paucimobilis (lane 2), S. multivorum (lane 5), S. spiritivorum (lane 9), and B. stolpii (lane 12); 3

lanes 3, 6, 10, and 13, the precipitates after centrifugation at 1000,000 $ g of the crude extracts 4

of S. paucimobilis (lane 3), S. multivorum (lane 6), S. spiritivorum (lane 10), and B. stolpii (lane 5

13); lanes 4, 7, 11, and 14, the supernatants after centrifugation at 1000,000 $ g of the crude 6

extracts of S. paucimobilis (lane 4), S. multivorum (lane 7), S. spiritivorum (lane 11), and B. 7

stolpii (lane 14). 8

9

FIG. 2. Sequence alignment and molecular phylogenetic tree of SPTs. Panel A, Aligned 10

sequences of bacterial SPTs, human SPT subunits (SPTLC1, SPTLC2 and SPTLC3), E. coli 11

AONS, and human ALAS2. The deduced amino acid sequences of the SPTs and other proteins 12

were aligned using the CLUSTALX version 1.83 program (68). The gap opening and extension 13

parameters were set to 10 and 0.2, respectively. Zymomonas SPT, Zymomonas mobilis SPT; 14

Sphingomonas SPT, Sphingomonas paucimobilis SPT; S. multivorum SPT, Sphingobacterium 15

multivorum SPT; S. spiritivorum SPT, Sphingobacterium spiritivorum SPT; Bdellovibrio SPT, 16

Bdellovibrio stolpii SPT. Residues identical among all the proteins are in light blue and those 17

conservatively substituted are in green. Residues indicated by the reversed triangle are active 18

site residues that are considered to be important for catalysis. The red-boxed sequences are the 19

SPT-specific PLP-binding motif (GTFSKSXXXXGG). Panel B, A molecular phylogenetic tree 20

of SPTs from various sources. The phylogenetic tree was constructed by the neighbor-joining 21

(NJ) method using the E. coli AONS protein as an outgroup. The number at each node 22

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represents the bootstrap value as a percentage of 1000 replications. 1

2

FIG. 3. Absorption spectra of purified SPTs. The purified SPTs (10 µM) were dissolved in 50 3

mM HEPES-NaOH (pH 7.5) containing 150 mM KCl and 0.1 mM EDTA, and their absorption 4

spectra were measured at 25˚C. Panels A-C, Absorption spectra of the Sphingomonas 5

paucimobilis, S. multivorum, and S. spiritivorum SPTs in the absence (solid line) and presence 6

(dashed-line) of 45 mM L-serine. Panel D, Absorption spectra of the B. stolpii SPT in the 7

absence (solid line) and presence (dashed-line) of 100 mM L-serine. The dotted line shows the 8

spectrum in the presence of 100 mM L-serine and 90 µM palmitoyl CoA. 9

10

FIG. 4. HPLC/ESI-ion-trap mass spectroscopy of reaction products of bacterial SPTs. Panel A, 11

ion chromatography of the reaction product of bacterial SPTs. Panel B, the structure of KDS and 12

the position of fragmentation and the size of the fragment ion are indicated. Panel C, MS data of 13

the reaction product of the S. multivorum SPT. Panel D, MS-MS data of the reaction product of 14

the S. multivorum SPT. 15

16

FIG. 5. HPLC/ESI-ion-trap mass spectroscopy of reaction products with various chain lengths 17

formed by the S. multivorum SPT. Panel A, ion chromatography of the reaction products with 18

various chain lengths formed by the S. multivorum SPT. Panel B, MS data of the reaction 19

product of the S. multivorum SPT from L-serine and arachidoyl CoA (C20:0). Panel D, MS-MS 20

data of the reaction product of the S. multivorum SPT from L-serine and arachidoyl CoA 21

(C20:0). 22

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1

FIG. 6. Kinetic characterization of recombinant SPTs. The enzyme assay was performed as 2

previously described (25). The apparent rate constants (kapp = v/[Et]) for the KDS formation 3

were plotted as a function of the palmitoyl CoA concentration at fixed concentrations of serine: 4 4

mM (open circle), 10 mM (open triangle), 20 mM (inverted open triangle), and 40 mM (open 5

square). Each solid line represents the theoretical curve according to the initial velocity kinetics 6

for the Ordered Bi-Bi mechanism using the kinetic parameters summarized in Table 2. Panel A, 7

S. multivorum SPT; Panel B, S. spiritivorum SPT; Panel C, B. stolpii SPT. 8

9

FIG. 7. Specificity of anti-SPT antibodies. The specificity of the antibodies against each 10

bacterial SPT were confirmed by Western blot analysis with cell lysates: lanes 1, 4, 7, E. coli 11

BL21(DE3) pLysS pET21 (empty vector); lane 2, E. coli BL21(DE3)pLysSpET21-aSPT 12

(expressing the Sphingomonas SPT); lane 3, Sphingomonas paucimobilis; lane 5, E. coli 13

BL21(DE3) pLysS pET21-bSPT (expressing the S. multivorum SPT); lane 6, S. multivorum; lane 14

8, E. coli BL21(DE3) pLysS pET21-dSPT (expressing the B. stolpii SPT); lane 9, B. stolpii. 15

16

FIG. 8. Electron microscopy of sphingolipid-containing bacteria. Upper Panels, morphological 17

examination of sphingolipid-containing bacteria. Ultrathin sections of the bacterial cells were 18

negatively stained and examined by electron microscopy. Panel A, Sphingomonas paucimobilis; 19

Panel B, S. multivorum; Panel C, B. stolpii. Middle Panels, the intracellular localization of SPT. 20

The localization of SPT was analyzed by the postembedding immunogold-labeling method of 21

electron microscopy. Ultrathin sections of bacterial cells were treated with anti-SPT antibody 22

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and stained with gold-labeled secondary antibodies. Panel D, Sphingomonas paucimobilis; 1

Panel E, S. multivorum; Panel F, B. stolpii. Arrows indicate the condensed distribution of 2

immunogold particles. Lower Panels, the negative control section. Ultrathin sections of 3

bacterial cells were treated with a preabsorbed primary antibody solution and stained with 4

gold-labeled secondary antibodies. An excess of the purified SPT protein was added to the 5

primary antibody working solution, and the solution was incubated at 4 °C for 48 hr before it 6

was added to each sections. Panel G, Sphingomonas paucimobilis; Panel H, S. multivorum; 7

Panel I, B. stolpii. Size markers are indicated. 8

9

10

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Table 1. Bacterial SPTs 1

2

Bacterial strain GenBank ORF [G+C] Mr pI 3

Accession No. (bp) (%) 4

S. paucimobilis AB055142 1,263 65.00 45,041 5.66 5

S. multivorum AB259214 1,200 38.33 43,771 5.05 6

S. spiritivorum AB259215 1,200 41.08 43,929 4.97 7

B. stolpii AB259216 1,263 43.31 46,172 6.25 8

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Table 2. Kinetic Parameters of bacterial SPTs 1

2

Bacterial strain Kd (Ser) Km (Ser) Km (Palmitoyl CoA) kcat 3

(mM) (mM) (mM) (s-1

) 4

S. paucimobilis 1.40 ± 0.10 4.7 ± 0.6 0.69 ± 0.09 2.3 ± 0.11 5

S. multivorum 0.47 ± 0.10 4.8 ± 0.6 0.10 ± 0.01 0.12 ± 0.01 6

S. spiritivorum 1.20 ± 0.03 5.0 ± 0.8 0.39 ± 0.04 0.15 ± 0.01 7

B. stolpii 2.55 ± 0.12 3.7 ± 0.4 ' 0.03 ± 0.002a 8

a v/[Et] value in the presence of 100 µM of palmitoyl CoA 9

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Figure.1 Ikushiro et al

KDS

Origin

Front

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Sphingomonas SPT -----------------------------------------------------------------------------------------------------------------MTEAAAQPHALP 12 Zymomonas SPT ----------------------------------------------------------------------------------------------------------------------------- 0 S.multivorum SPT ----------------------------------------------------------------------------------------------------------------------------- 0 S.spiritivorum SPT ----------------------------------------------------------------------------------------------------------------------------- 0 Bdellovibrio SPT ----------------------------------------------------------------------------------------------------------------------------M 1 Human SPTLC2 -----------------------MRPEPGGCCCRRTVRANGCVANGEVRNGYVRSSAAAAAAAAAGQIHHVTQNGG---LYKRPFNEAFEETPMLVAVLTYVGYGVLTLFGYLRDFLRYWRIEKC 99 Human SPTLC3 ------------------------MANPGG----------GAVCNGKLHNHKKQSNGSQSRNCTKNGIVKEAQQNGKPHFYDKLIVESFEEAPLHVMVFTYMGYGIGTLFGYLRDFLRNWGIEKC 91 Human SPTLC1 ----------------------------------------------------------------------------MATVTEQWVLVEMVQALYEAPAYHLILEGILILWIIRLLFSKTYKLQER 49 E.coli AONS ----------------------------------------------------------------------------------------------------------------------------- 0 Human ALAS2 MVTAAMLLQCCPVLARGPTSLLGKVVKTHQFLFGIGRCPILATQGPNCSQIHLKATKAGGDSPSWAKGHCPFMLSELQDGKSKIVQKAAPEVQEDVKAFKTDLPSSLVSVSLRKPFSGPQEQEQI 125 Sphingomonas SPT ADAPDIAPERDLLSKFDG-----------LIAERQKLLDSGVTDPFAIVMEQVKSPTEAVIRG-----KDTILLGTYNYMGMTFDP-DVIAAGKEALEKFGSGTNGSRMLNGTFHDHMEVEQALR 120 Zymomonas SPT ------MQVTDLFSKFDP-----------LIAIRKEMLSHGGRDPFAVVMEKVLSPTKAIIKG-----KPVILLGTYNYMGMTFDP-DVIAAGEKALKEFGAGTTGSRVLNGTYQGHKACEEALK 102 S.multivorum SPT ---MSKGKLGEKISQFK-------------IVE---ELKAKGLYAYFRPIQSKQD-TEVKIDG-----RRVLMFGSNSYLGLTTDT-RIIKAAQDALEKYGTGCAGSRFLNGTLDIHVELEEKLS 99 S.spiritivorum SPT ---MSKGKLSERISHFN-------------IVE---ELKSKGLYAYFRPIQSKQD-TEVMIDG-----KRVLMFGSNSYLGLTIDP-RIIEAAQDALSKYGTGCAGSRFLNGTLDIHIELEHKLS 99 Bdellovibrio SPT KHNLQDNLQGEQMANTNSNGGKKPF-SDAKIIERANLLRDNDLYFFFRAIEETEA-STVTVKG-----KKQIMIGSNNYLGLTHHP-AVKEAAIKAVEKYGTGCTGSRFLNGNLNIHEELDEKLA 118 Human SPTLC2 HHATEREEQKDFVSLYQDFENFYTRNLYMRIRDNWNRPICSVPGARVDIMERQSHDYNWSFKYTGNIIKGVINMGSYNYLGFARNTGSCQEAAAKVLEEYGAGVCSTRQEIGNLDKHEELEELVA 224 Human SPTLC3 NAAVERKEQKDFVPLYQDFENFYTRNLYMRIRDNWNRPICSAPGPLFDLMERVSDDYNWTFRFTGRVIKDVINMGSYNFLGLAAKYDESMRTIKDVLEVYGTGVASTRHEMGTLDKHKELEDLVA 216 Human SPTLC1 SD-LTVKEKEELIEEWQP--------------EPLVPPVPKNHPALNYNIESGPPSHNTVVNG-----KECINFASFNFLGLLDNP-RVKAAALASLKKYGVGTCGPRGFYGTFDVHLDLEERLA 153 E.coli AONS -----MSWQEKINAALD---------------------ARGAADALRRRYPVAQGAGRWLVADD----RQYLNFSSNDYLGLSHHP-QIIRAWQQGAEQFGIGSGGSGHVSGYSVVHQALEEELA 94 Human ALAS2 SGKVTHLIQNNMPGNYVFSYDQFFRDKIMEKKQDHTYRVFKTVNRWADAYPFAQHFSEASVAS-----KDVSVWCSNDYLGMSRHP-QVLQATQETLQRHGAGAGGTRNISGTSKFHVELEQELA 244 Sphingomonas SPT DFYGTTGAIVFSTGYMANLGIISTLAGKGE--YVILDADSHASIYDGCQQGNAEIVRFRHNSVEDLDKRLGR-LPKEP---------AKLVVLEGVYSMLGDIAPLKEMVAVAKKHGAMVLVDEA 233 Zymomonas SPT DYYGMEHAMVFSTGYQANLGMISTLAGKGE--YVIIDADSHASIYDGCRLGNAEIIRFRHNSPEDLDRRLAR-LPKEA---------GKLVVLEGVYSMLGDIAPLAEMVAIAKKHDALILDDEA 215 S.multivorum SPT AYVGKEAAILFSTGFQSNLGPLSCLMGRND--YILLDERDHASIIDGSRLSFSKVIKYGHNNMEDLRAKLSR-LPEDS---------AKLICTDGIFSMEGDIVNLPELTSIANEFDAAVMVDDA 212 S.spiritivorum SPT QLVGKEASILFSTGFQSNLGPISCLMGRND--YILLDERDHASIIDGSRLSFSKVIKYGHNDMDDLRAKLSR-LPSES---------AKLIVTDGIFSMEGDIVNLPEMVKIADEYDAALMVDDA 212 Bdellovibrio SPT AYLGHEKAIVFSTGMQANLGALSAICGPKD--LMLFDSENHASIIDASRLSLGTTFKYKHNDMASLEELLESNMSRFN---------RVIIVADGVFSMTGDILRLPEVVKLAKKYGAYVYVDDA 232 Human SPTLC2 RFLGVEAAMAYGMGFATNSMNIPALVGKGC--LILSDELNHASLVLGARLSGATIRIFKHNNMQSLEKLLKDAIVYGQPRTRRP-WKKILILVEGIYSMEGSIVRLPEVIALKKKYKAYLYLDEA 346 Human SPTLC3 KFLNVEAAMVFGMGFATNSMNIPALVGKGC--LILSDELNHTSLVLGARLSGATIRIFKHNNTQSLEKLLRDAVIYGQPRTRRA-WKKILILVEGVYSMEGSIVHLPQIIALKKKYKAYLYIDEA 338 Human SPTLC1 KFMKTEEAIIYTYGFATIASAIPAYSKRGD--IIFVDRAACFAIQKGLQASRSDIKLFKHNDMADLERLLKEQEIEDQKNPRKARVTRRFIVVEGLYMNTGTICPLPELVKLKYKYKARIFLEES 276 E.coli AONS EWLGYSRALLFISGFAANQAVIAAMMAKED--RIAADRLSHASLLEAASLSPSQLRRFAHNDVTHLARLLASPCPGQQ-----------MVVTEGVFSMDGDSAPLAEIQQVTQQHNGWLMVDDA 206 Human ALAS2 ELHQKDSALLFSSCFVANDSTLFTLAKILPGCEIYSDAGNHASMIQGIRNSGAAKFVFRHNDPDHLKKLLEKSNPKIP----------KIVAFETVHSMDGAICPLEELCDVSHQYGALTFVDEV 359 Sphingomonas SPT HSMGFFGPNGRGVYEAQGLEG-QIDFVVGTFSKSVGTVGGFVVSNHPKFEAVRLACRPYIFTASLPPSVVATATTSIRKLMTAH------EKRERLWSNARALHGGLKAMGFRLGTETCDSAIVA 351 Zymomonas SPT HGMGFFGKNGRGVFEELGLEG-QIDFIVGTFSKSVGTVGGFCVSNHPQFEVLRLVCRPYVFTASLPPSVVATAEASIRKLQKAN------DKREHLWKNSRRLHGGLKEMGFKLGTETAQSAIIA 333 S.multivorum SPT HSLGVIGHKGAGTASHFGLND-DVDLIMGTFSKSLASLGGFVAGDADVIDFLKHNARSVMFSASMTPASVASTLKALEIIQNEP------EHIEKLWKNTDYAKAQLLDHGFDLG--ATESPILP 328 S.spiritivorum SPT HSLGVIGEHGAGTASHFGLTD-KVDLIMGTFSKSLASLGGFVAGDADVIDYLKHNARSVMFSASMTPASVASTLKALEIMISEP------EHMENLWKNTNYAKQQLLESGFDLG--ATESPILP 328 Bdellovibrio SPT HGLGVMGPQGRGTMAHFDVTK-DVDFNMGTFSKSFASIGGVISGSKDAIDYVRHSARSFMFSASMPPAAVATVSACIDVVQNDE------TILNNLWSNVEFMRNGFKELGFFTY--GSQTPIIP 348 Human SPTLC2 HSIGALGPTGRGVVEYFGLDPEDVDVMMGTFTKSFGASGGYIGGKKELIDYLRTHSHSAVYATSLSPPVVEQIITSMKCIMGQDGTSLGKECVQQLAENTRYFRRRLKEMGFIIYG-NEDSPVVP 470 Human SPTLC3 HSIGAVGPTGRGVTEFFGLDPHEVDVLMGTFTKSFGASGGYIAGRKDLVDYLRVHSHSAVYASSMSPPIAEQIIRSLKLIMGLDGTTQGLQRVQQLAKNTRYFRQRLQEMGFIIYG-NENASVVP 462 Human SPTLC1 LSFGVLGEHGRGVTEHYGINIDDIDLISANMENALASVGGFCCGRSFVIDHQRLSGQGYCFSASLPPLLAAAAIEALNIMEENP------GIFAVLKEKCGQIHKSLQGISGLKVVGESLSPAFH 395 E.coli AONS HGTGVIGEQGRGSCWLQKVKP---ELLVVTFGKGFGVSGAAVLCSSTVADYLLQFARHLIYSTSMPPAQAQALRASLAVIRSDEGD----ARREKLAALITRFRAGVQDLPFTLAD--SCSAIQP 322 Human ALAS2 HAVGLYGSRGAGIGERDGIMH-KIDIISGTLGKAFGCVGGYIASTRDLVDMVRSYAAGFIFTTSLPPMVLSGALESVRLLKGEEGQ----ALRRAHQRNVKHMRQLLMDRGLPVIP--CPSHIIP 477 Sphingomonas SPT VMLEDQE----QAAMMWQALL----DGGLYVNMARPPATPA---GTFLLRCSICAEHTPAQIQTVLGMFQAAGRAVGVIG---------------------------------------- 420 Zymomonas SPT VILPDQE----QAVAMWQNLL----ELGLYVNLARPPATPA---GMFLLRCSLCAEHSDDDVTEILAMFKKAGQATGVLAA--------------------------------------- 403 S.multivorum SPT IFIRSNE----KTFWVTKMLQ----DDGVFVNPVVSPAVPA---EESLIRFSLMATHTYDQIDEAIEKMVKVFKQAEVETLI-------------------------------------- 399 S.spiritivorum SPT IFIRNNE----KTFWVTKMLQ----DDGVFVNPVVSPAVPS---EESLIRFSLMATHTFDQIDEAVEKMVRVFKQAEIESLI-------------------------------------- 399 Bdellovibrio SPT LFIGDDM----KALKMTKWLE----SKGVFCTPVLPPAVPK---GETLIRTSYMASHNREDLSTVLEVFAEAKKIFDIPNHLH------------------------------------- 420 Human SPTLC2 LMLYMPA----KIGAFGREML----KRNIGVVVVGFPATPI---IESRARFCLSAAHTKEILDTALKEIDEVGDLLQLKYSRHRLVPLLDRPFDETTYEETED----------------- 562 Human SPTLC3 LLLYMPG----KVAAFARHML----EKKIGVVVVGFPATPL---AEARARFCVSAAHTREMLDTVLEALDEMGDLLQLKYSRHKKS-ARPELYDETSFELED------------------ 552 Human SPTLC1 LQLEESTGSREQDVRLLQEIVDQCMNRSIALTQARYLEKEEKCLPPPSIRVVVTVEQTEEELERAASTIKEVAQAVLL------------------------------------------ 473 E.coli AONS LIVGDNS----RALQLAEKLR----QQGCWVTGIRPPTVPA---GIARLRLTLTAAHEMQDIDRLLEVLHGNG----------------------------------------------- 384 Human ALAS2 IRVGNAA----LNSKLCDLLLS---KHGIYVQAINYPTVPR---GEELLRLAPSPHHSPQMMEDFVEKLLLAWTAVGLPLQDVSVAACNFCRRPVHFELMSEWERSYFGNMGPQYVTTYA 557

Figure 2A Ikushior et al.

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Figure 2B Ikushiro et al

0.05E. coli AONS

Sphingomonas SPT

Zymomonas SPT

Bdellovibrio SPT

S. spiritivorum SPT

S. multivorum SPT

S. cerevisiae LCB1

Mouse LCB1

Human SPTLC1

S. ceravisiae LCB2

Human SPTLC3

Mouse LCB2

Human SPTLC2

100

100

100

100

100

10082

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100

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20

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0

A

30

20

10

0500400300

C

30

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0500400300

D

30

20

10

0

B

Figure3 Ikushiro et al

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0

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Wavelength [nm] Wavelength [nm]

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m/z

282

270

300264

Figure 4 Ikushiro et al

A

270

–HCHO

+OH

O

NH3

3

282

–H2O

3-Ketodihydrosphingosine (KDS)

B

C DRetention Time (min)

Rela

tive A

bund

ance

0 10 20 30

0

50

100

S.multivorum

S.spiritivorum

B.stolpii

Sphingomonas

300

200 250 300 350m/z

Re

lative

Abu

nda

nce

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364

226

Re

lative

Ab

un

da

nce

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100 200 300150 250

254.4240.495.0 301.2109.1 135.1149.1 165.0 221.1184.1

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250 300 350

m/z

0

20

40

60

80

100

Rela

tive A

bundance

356.4

357.4


0 10 20

Retention Time (min)

30

12:0-CoA

14:0-CoA

14:1-CoA

16:0-CoA

16:1-CoA

18:0-CoA

18:1-CoA

20:0-CoA

Substrates

244

272

270

300

298

328

326

356

0

50

100

Rela

tive A

bun

dance

Products

m/z

A B

C

100 150 200 250 300 350

m/z

–H2O

338

339.2

310.6357.2

320

–2H2O

326

–HCHO

0

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Re

lative

Ab

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0210

Palmitoyl CoA [mM]

5

0

ka

pp [ m

in -1

]

5

0

Figure 6. Ikushiro et al

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B

C

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83

62

48

33

25

kDa

1 2 3 4 5 6 7 8 9

Fig.7 Ikushiro et al

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400 nm

400 nm

400 nm

400 nm

200 nm

400 nm

200 nm

200 nm

200 nm

A B C

D E F

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