Sv - jbc.org€¦ · 19/07/2019 · S-13 Figure S6: Investigation of Cellvibrio japonicus GH74...
Transcript of Sv - jbc.org€¦ · 19/07/2019 · S-13 Figure S6: Investigation of Cellvibrio japonicus GH74...
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Substrate specificity, regiospecificity and processivity in Glycoside Hydrolase family 74
Gregory Arnal1, Peter J. Stogios2, Jathavan Asohan1, Mohamed Attia1, Tatiana Skarina2, Alexander Holm
Viborg1, Bernard Henrissat3,4, Alexei Savchenko2,5,*, Harry Brumer,1,*.
List of the material included:
Table S1: Primers used for the PCR amplification of GH74 modules encoding sequence.
Table S2: Primers used for the site-directed mutagenesis of PoGH74cat and CjGH74.
Table S3: X-ray crystallographic statistics.
Figure S1: pH profiles.
Figure S2: temperature profiles
Figure S3: Michael Menten kinetics
Figure S4: Time course analysis of tamarind XyG hydrolysis in early stage of the reaction by
HPAEC-PAD
Figure S5: Protein sequence alignment of PoGH74, PgGH74 and SrGh74 from group 5,
SvGH74b from group 4, SatGH74 from Group3 and ClGH74a and NkGH74 from Group1.
Figure S6: Investigation of Cellvibrio japonicus GH74 mode of action.
Figure S7. Regiospecificity of PoGH74 wild type, of ΔAsn642-Asn651 variant and of different
point mutations of residue G442 located in the (-1) subsite of the active cleft of PoGH74.
Figure S8: Details of active sites of PgGH74 and PoGH74cat•(XXLG+XGXXLG) (PDB 6MGL)
crystal structures.
Supplementary file: GH74-bioinformatics analysis.mfa. This file contains the alignment used for
generating the phylogenetic tree in the Figure 1 in the main text. It contains all the non-redundant
sequences encoding for GH74 catalytic modules referenced in the CAZy database as well as the
sequence of the previously characterized GH74 modules from Xanthomonas citri pv.
mangiferaeindicae (Genbank accession number CCG35167) and Aspergillus fumigatus (Genbank
XP_747057), and the sequence of three uncharacterized GH74 modules from Paenibacillus
polymyxa Sb3-1, Paenibacillus jamilae and Streptomyces atroolivaceus (Genbank
WP_019687396, WP_063210590 and WP_033303664, respectively) that were recombinantly
produced in this study.
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Table S1: Primers used for the PCR amplification of GH74 modules encoding sequence. LIC-compatible extensions
TACTTCCAATCCAATGCCATG and TTATCCACTTCCAATGTTA were added at the 5’ extremity of each forward and reverse primers,
respectively.
Protein
(GenBank)
Bacterial strain
(DSM-No) Primer forward Primer reverse
Cloning vector-
N-terminal protein tag
ClGH74a
(AEM74844)
Caldicellulosiruptor lactoaceticus
(DSM 9545)
GCAGCGTCGGAACCATAC
ACATGG
AGAGGCTGTTGGTGTTGGC
AGTCC pMCSG53-His
ClGH74b
(AEM72711)
Caldicellulosiruptor lactoaceticus
(DSM 9545)
GCTGAAGTTCTTTCACAAA
AATATGTATGGAAGAATG
TGTTACTGTTCCACCACTTC
CACTATCAATTTC pMCSG69-MBP
CbGH74
(ACM60948)
Caldicellulosiruptor bescii
(DSM 6725)
AGTATAGTCACCACTCAAA
AATACATTTGGAAAAATG
TGATGTTGGAATTGTAGCA
CTATCAGCAATTTC pMCSG69-MBP
NkGH74
(AEV98114)
Niastella koreensis GR20-10
(DSM 17620)
ACAAAACTAATTGCACAA
ACATTTGGTAATGTAGCC
ATGGGTAAATTGAAGGGG
CAGCGC pMCSG53-His
PgGH74
(AIQ68396)
Paenibacillus graminis
(DSM 15220)
AGCGAAGCCTATAACTGG
AAAAGTGTCG
CACTGGATCGGCATAGAGC
GTGC pMCSG53-His
PbGH74
(AIQ58082)
Paenibacillus borealis
(DSM 13188)
AGTGAAGCCTACAACTGG
AACAGCG
TACCGGATCGGCGTATAAG
GTACC pMCSG53-His
PpGH74
(WP_019687396)
Paenibacillus polymyxa
(DSM 36)
GCTCCAAGTGACGATTATA
CTTGGAAAAG
TTACACACGATCTGCCACC
AGAATGCC pMCSG53-His
PjGH74
(WP_063210590)
Paenibacillus jamilae
(DSM13815)
GCTCCGAGTGACGATTATA
CTTGGAAAAG
TTATACACGATCTGCCACC
AGAATGCC pMCSG53-His
PmGH74
(AFC30493)
Paenibacillus mucilaginosus
(synthetic gene)
GTGAAAACACAAGCGTAC
GACTGGG
ATCCTTCATGTCCCCGCGA
ACG pMCSG53-His
RaGH74a
(ADU23213)
Ruminococcus albus 7
(DSM 20455)
GCTCAGGATATTTCATCGT
CTATGGAATGG
TGTTATCTTGGGAGCCTTG
ATATCTCCG pMCSG-GST
RaGH74b
(ADU20593)
Ruminococcus albus 7
(DSM 20455)
TCTGTTGATACGGGCTGGG
AATGG
TCACTTAACATCTCCACGG
ATTATACCGC pMCSG53-His
AFV00434 Simiduia agarivorans SA1
(DSM 21679)
ATGGCCGCTACCGTATGGC
AGC
CGCCGGATCCATATTCATG
CCGC pMCSG69-MBP
AFV00474 Simiduia agarivorans SA1
(DSM 21679)
ACACCCAATTGGGAATCAC
TCAATCCC
CGACAGACCATCAGGCGCC
G pMCSG69-MBP
SrGH74
(AGP53770)
Streptomyces rapamycinicus
(DSM 41530)
GCCGACGCCTACACCTGGA
AG
TCAGGAGATGTCGCCGCGC
AG pMCSG53-His
SvGH74a
(CCA60033)
Streptomyces venezuelae
(DSM 40230) GCGGCGACCGGCCCGTAC
TCAGGACGGGTCGCCGTAC
TG pMCSG53-His
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SvGH74b
(CCA55868)
Streptomyces venezuelae
(DSM 40230)
CCCTCCTACACCTGGAAGA
ACGTC CTGCTCGGCCGTGTCGCC pMCSG53-His
SatGH74
(WP_033303664)
Streptomyces atroolivaceus
(DSM 40137)
GCGAAGACCCAGCCGCAC
CACTGG
CTACTGGGGGTCTGCGTAC
TGG pMCSG53-His
Table S2: Primers used for the site-directed mutagenesis of PoGH74cat and CjGH74. Bold characters indicate point mutations.
Protein Matrix DNA Primer forward Primer reverse Reference
PoGH74cat-G476A pMCSG53::PoGH74cat CACTGCACTTGCCGACGT
TTCCGGTTTC
GAAACCGGAAACGTCGG
CAAGTGCAGTG
Arnal et al.
PoGH74cat-G476Q pMCSG53::PoGH74cat CACTGCACTTCAAGACGT
TTCCGGTTTC
GAAACCGGAAACGTCTT
GAAGTGCAGTG
Arnal et al.
PoGH74cat-G476W pMCSG53::PoGH74cat CACTGCACTTTGGGACGT
TTCCGGTTTC
GAAACCGGAAACGTCCC
TAAGTGCAGTG
Arnal et al.
CjGH74-W353A pET28a::CjGH74 GCGTCGCAAATTGCCGCG
TGGCCCGATGCGGTT
AACCGCATCGGGCCACG
CGGCAATTTGCGACGC
Attia et al.
CjGH74-W354A pET28a::CjGH74 TCGCAAATTGCCTGGGCG
CCCGATGCGGTTTTC
GAAAACCGCATCGGGCG
CCCAGGCAATTTGCGA
Attia et al.
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Table S3: X-ray crystallographic statistics
Structure AFV00434 NkGH74•(XXLG/XXXG) ClGH74a•(LLG) PgGH74 SrGH74•(XLLG/XXXG)
PDB code 6P2K 6P2L 6P2M 6P2N 6P2O
Data collection
Space group P213 P22121 P212121 P212121 P212121
Unit cell a, b, c (Å) α, β, γ, (°)
173.4, 173.4, 173.4 90, 90, 90
42.9, 119.1, 127.0 90, 90, 90
65.0, 72.8, 150.7 90, 90, 90
57.5, 86.2, 130.2 90, 90, 90
93.0, 112.8, 222.8 90, 90, 90
Resolution, Å 40.0 – 2.15 30.0 – 1.08 25.00 – 1.98 35.00 – 1.35 25.00 – 1.88 Rmerge
a
Rpimb
0.072 (0.849)c 0.032 (0.441)
0.073 (0.825) 0.037 (0.447)
0.165 (0.873) 0.063 (0.438)
0.049 (0.838) 0.021 (0.449)
0.082 (0.954) 0.043 (0.565)
CC1/2c 0.676 0.605 0.623 0.567 0.541
I / (I) 22.2 (1.8) 25.9 (1.7) 18.2 (2.4) 31.0 (1.7) 12.2 (1.1)
Completeness, % 99.8 (97.5) 98.3 (96.0) 100 (99.9) 96.1 (96.8) 89.4 (90.1) Redundancy 5.7 (4.6) 4.5 (4.1) 7.8 (6.3) 6.1 (4.2) 3.7 (3.6) Refinement Resolution, Å 38.78 – 2.15 29.3 – 1.08 24.6 – 1.98 30.5 – 1.35 25.0 – 1.88 No. unique reflections: working, test
93430, 4654 262889, 1916 47656, 1875 132023, 3636
156964, 3304
R-factor/free R-factord 15.4/20.4 (23.8/27.4)
11.9/14.0 (23.9/24.5) 15.6/20.4 (23.8/25.6)
14.9/18.0 (22.9/20.8)
14.8/18.0 (28.9/30.4)
No. refined atoms Protein Carbohydrate Solvent Water
12080 N/A 150 1794
10165 155 27 1562
5294 74 45 1229
5724 N/A 21 1339
10995 381 40 2662
B-factors Protein Carbohydrate Solvent Water
33.8 N/A 68.8 44.3
11.4 22.7 17.6 31.8
23.3 47.5 52.1 39.8
17.3 N/A 34.2 33.4
23.3 35.6 66.8 41.6
r.m.s.d. Bond lengths, Å
Bond angles,
0.004 0.663
0.008 1.112
0.003 0.572
0.013 1.289
0.013 1.133
Ramachandran favoured 1501/1556 (96%) 690/711 (97%) 682/700 (97%) 731/761 (96%) 1402/1460 (96%) allowed 53/1556 (3%) 21/711 (3%) 18/700 (3%) 30/761 (4%) 58/1460 (4%) outliers 2/1556 (0%) 0 0 0 0
aRmerge = hklj|Ihkl.j - Ihkl|/hkljIhk,j, where Ihkl,j and Ihkl are the jth and mean measurement of the
intensity of reflection j. bRpim = hkl√(n/n-1) n
j=1|Ihkl.j - Ihkl|/hkljIhk,j call values in brackets and CC1/2 refer to the highest resolution shell. dR = |Fp
obs – Fpcalc|/Fp
obs, where Fpobs and Fp
calc are the observed and calculated structure factor
amplitudes, respectively.
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Figure S1: pH profiles for A-NkGH74 (Group 1), B-RaGH74a (Group 1), C-ClGH74a (Group 1), D-
SatGH74 (Group 3), E-SvGH74a (Group 4), F-ClGH74b (Group 5), G-CbGH74 (Group 5), H-RaGH74b
(Group 5), I-PmGH74 (Group 5), J-PgGH74 (Group 5), K-PbGH74 (Group 5), L-PjGH74 (Group 5), M-
PpGH74 (Group 5), N-SrGH74 (Group 5) and O-SvGH74b (Group 5). Blue circles for citrate buffer; Green
crosses for phosphate buffer. Error bars represent the standard deviation for three replicates.
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Figure S2: temperature profiles for A-NkGH74 (Group 1), B-RaGH74a (Group 1), C-ClGH74a (Group
1), D-SatGH74 (Group 3), E-SvGH74a (Group 4), F-ClGH74b (Group 5), G-CbGH74 (Group 5), H-
RaGH74b (Group 5), I-PmGH74 (Group 5), J-PgGH74 (Group 5), K-PbGH74 (Group 5), L-PjGH74
(Group 5), M-PpGH74 (Group 5), N-SrGH74 (Group 5) and O-SvGH74b (Group 5). Error bars represent
the standard deviation for three replicates.
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Figure S3: Michael Menten kinetics for A-NkGH74 (Group 1), B-RaGH74a (Group 1), C-ClGH74a
(Group 1), D-SatGH74 (Group 3), E-SvGH74a (Group 4), F-ClGH74b (Group 5), G-CbGH74 (Group 5),
H-RaGH74b (Group 5), I-PmGH74 (Group 5), J-PgGH74 (Group 5), K-PbGH74 (Group 5), L-PjGH74
(Group 5), M-PpGH74 (Group 5), N-SrGH74 (Group 5) and O-SvGH74b (Group 5). Error bars represent
the standard deviation for three replicates.
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Figure S4: Time course analysis of tamarind XyG hydrolysis in early stage of the reaction by
HPAEC-PAD. A-NkGH74 (Group 1), B-RaGH74a (Group 1), C-ClGH74a (Group 1), D-SatGH74
(Group 3), E-SvGH74a (Group 4), F-ClGH74b (Group 5), G-CbGH74 (Group 5), H-RaGH74b (Group 5),
I-PmGH74 (Group 5), J-PgGH74 (Group 5), K-PbGH74 (Group 5), L-PjGH74 (Group 5), M-PpGH74
(Group 5), N-SrGH74 (Group 5) and O-SvGH74b (Group 5)
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Figure S5: Protein sequence alignment of PoGH74, PgGH74 and SrGh74 from group 5, SvGH74b
from group 4, SatGH74 from Group3 and ClGH74a and NkGH74 from Group1. Stars indicate active cleft
hydrophobic residues and their subsite position. The color code of the stars corresponds to their
conservation across the phylogenetic groups: blue stars when the residue is only conserved in Group 5,
purple stars when the residue is conserved in Groups 4 and 5, green stars when the residue is conserved in
Groups 3, 4 and 5, red stars when the residue is conserved across all Groups. Red triangles indicate the
catalytic residues. Grey diamond refers to the residue in the -1 subsite dictating regiospecificity. Green
bars indicate specific loop extensions. Residue numbering is based on PoGH74 full-length protein.
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Figure S6: Investigation of Cellvibrio japonicus GH74 mode of action. HPAEC-PAD analysis of time-
course tamarind XyG hydrolysis by A. CjGH74 wild type, B. CjGH74-W353A variant (+3 subsite) and
C. CjGH74-W354A variant (+5 subsite). The same stock solution of tamarind XyG was used for each
variant, hence all “no enzyme” traces are identical. The same reference chromatogram of xyloglucan
oligosaccharide standards is presented for comparison with the products of each variant.
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Figure S7. Regiospecificity of PoGH74 wild type, of ΔAsn642-Asn651 variant and of different point
mutations of residue G442 located in the (-1) subsite of the active cleft of PoGH74. Limit digest of
(A) XXXGXXXG and (B) XXXG analyzed by HPAEC-PAD.
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Figure S8: Details of active sites of PgGH74 and PoGH74cat•(XXLG+XGXXLG) (PDB 6MGL) crystal
structures. Comparison of PgGH74 and PoGH74cat•(XXLG+XGXXLG) (PDB 6MGL), showing the
presence of 12 aromatic residues in the active site cleft of PgGH74 and conformational differences in the -
4, -3 and -2 subsites (i.e. different positions of W126, Y214 and Y122 of PgGH74 relative to their
equivalents in PoGH74 – W91, Y87, Y219).