THE JOURNAL OF BIOLOGICAL CHEMISTRY (c) 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 19, , Issue of July 5, pp. 12646-12654,1991 Printed in U.S.A.

&(L-cu-Aminoadipyl)-L-cysteinyl-D-valine Synthetase from Aspergillus nidulans MOLECULAR CHARACTERIZATION OF THE acuA GENE ENCODING THE FIRST ENZYME OF THE PENICILLIN BIOSYNTHETIC PATHWAY*

(Received for publication, November 16, 1991)

Andrew P. MacCabe$$, Henk van Liemptll, Harriet Palissall, Shiela E. UnklesS, Maureen B. R. Riach$II, Eva Pfeiferll, Hans von Dohrenll, and James R. Kinghorn$** From the $Molecular Genetics Unit, Sir Harold Mitchell Building, University of St. Andrews, St. Andrews, Fife KY16 9TH, United KinEdom and the Tlnstitut fur Biochemie und Molekulare Biologie, Technische Universitat Berlin, Franklinstrasse 29, 0-1000, Beylin, Germany

The Aspergillus nidulans gene (acvA) encoding the first catalytic steps of penicillin biosynthesis that re- sult in the formation of &(L-a-aminoadipy1)-L-cyste- inyl-D-valine (ACV), has been positively identified by matching a 15-amino acid segment of sequence ob- tained from an internal CNBr fragment of the purified amino-terminally blocked protein with that predicted from the DNA sequence. acvA is transcribed in the opposite orientation to ipnA (encoding isopenicillin N synthetase), with an intergenic region of 872 nucleo- tides. The gene has been completely sequenced at the nucleotide level and found to encode a protein of 3,770 amino acids (molecular mass, 422,486 Da). Both fast protein liquid chromatography and native gel esti- mates of molecular mass are consistent with this pre- dicted molecular weight. The enzyme was identified as a glycoprotein by means of affinity blotting with con- canavalin A. No evidence for the presence of introns within the acvA gene has been found.

The derived amino acid sequence of ACV synthetase (ACVS) contains three homologous regions of about 585 residues, each of which displays areas of similarity with (i) adenylate-forming enzymes such as parsley 4- coumarate-CoA ligase and firefly luciferase and (ii) several multienzyme peptide synthetases, including bacterial gramicidin S synthetase 1 and tyrocidine syn- thetase 1. Despite these similarities, conserved cys- teine residues found in the latter synthetases and

* The work at the Technical University of Berlin was supported by grants (to H. D.) from the Bundesministerium fur Forschung und Technology, Gist-brocades NV (Netherlands), and the Deutsche For- schungsgemeinschaft (Do 270/5-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTM/EMBL Data Bank with accession number(s) The nucleotide sequence(s) reported in thispaper has been submitted

X54853. Supported by the Maitland Ramsay Trust, University of St.

Andrews. Awarded a short term fellowship by the European Molecular Biology Organization, which facilitated the preparation and purifi- cation of ACVS (Grant ASTF 5936).

11 Awarded a biotechnology postgraduate studentship award by the Science and Engineering Research Council, Biotechnology Director- ate “Antibiotics Initiative.”

** Supported by grants GR/D/47093 and GR/F/5591 from the Science and Engineering Research Council, Biotechnology Director- ate “Antibiotics Initiative.” T o whom correspondence should be ad- dressed Molecular Genetics Unit, Sir Harold Mitchell Building, University of St. Andrews, St. Andrews, Fife KY16 9TH, United Kingdom. Tel.: 0334-76161 (ext. 7156); Fax: 0334-78721.

thought to be essential for the thiotemplate mechanism of peptide biosynthesis have not been detected in the ACVS sequence. These observations, together with the occurrence of putative 4’-phosphopantetheine-attach- ment sites and a putative thioesterase site, are dis- cussed with reference to the reaction sequence leading to production of the ACV tripeptide. We speculate that each of the homologous regions corresponds to a func- tional domain that recognizes one of the three substrate amino acids.

The structural genes encoding the second (isopenicillin N synthetase) and final (penicillin acyltransferase) enzymes required for penicillin biosynthesis have been isolated and characterized at the nucleotide level in a number of microor- ganisms (1-7). Recent work has provided evidence for the existence of a penicillin gene cluster in the filamentous fungi Aspergillus nidulans and Penicillium chrysogenum (8,9). The A. nidulans gene cluster was found to correspond to npeA of A. nidulans (8,9), a locus previously associated with penicillin biosynthesis by virtue of loss-of-function mutations (10, 11). That the essential catalytic machinery for penicillin biosyn- thesis is encoded within this region was demonstrated by transgenic expression of the cluster in penicillin nonproducing organisms (12).

Previous evidence from experiments using cell-free extracts has suggested that either two separate enzymes are involved in the synthesis of the 6-(L-cY-aminoadipyl)-L-cysteinyl-D- valine (ACV)’ tripeptide from the component amino acids (13, 14) or that a single multifunctional enzyme is required (15, 16). Recently the A. nidulans ACVS protein has been purified to homogeneity and shown to be a single polypeptide unit of apparent molecular mass of 230,000 Da (17). It has been demonstrated that this multienzyme catalyzes the for- mation of ACV from the three constituent amino acids and ATP, and a reaction sequence according to the thiotemplate mechanism (18,19) has been proposed.

We report here the conclusive identification of the gene encoding the ACVS of A. nidulans, further characterization of the protein, and the alignment of additional sequence data to ensure correspondence of gene and gene product.

’ The abbreviations used are: ACV, 6-(L-a-aminoadipy1)-L-cystei- nyl-D-valine; ACVS, ACV synthetase; ORF, open reading frame; FPLC, fast protein liquid chromatography; SDS, sodium dodecyl sulfate; nt, nucleotide(s).

12646

A. nidulans ACV Synthetase 12647

EXPERIMENTAL PROCEDURES

Media and Strains-Escherichia coli strain DH5 (F-, recAl, endAl, hsdRl7 (rk-, mk+) supE44, X-, thi-1, gyrA96, relAl) was used for the propagation of all plasmids and the preparation of competent cells (20).

DNA Manipulations-Ail standard DNA techniques employed in the cloning and generation of recombinant plasmids were as detailed in Ref. 20. Plasmid preparation was carried out by alkaline lysis and CsCl purification (20). Nucleotide sequences of DNA fragments sub- cloned into pUC vectors from pSTA201 and pSTA207 were deter- mined using the dideoxynucleotide chain termination procedure (21) on purified double-stranded plasmids. As the open reading frame was found to continue beyond the recombinant DNA fragment cloned into pSTA207, a subclone (pSTA230) of cosmid CX35 (12) was used to extend the sequence. 17-mer oligonucleotide primers, purchased from Dr. A. Hawkins (Department of Biochemistry and Genetics, University of Newcastle, Newcastle, United Kingdom and the De- partment of Biochemistry and Microbiology, University of St. An- drews, St. Andrews, United Kingdom) were used to facilitate the sequencing of DNA stretches.

Transcript Mapping-Primer extension was performed according to the method of Williams and Mason (22). Total RNA (100 pg) isolated as described by MacCabe et al. (8) from mycelium grown under induced conditions, i.e. in fermentation medium, was hybrid- ized overnight a t 60 "C to 2.5 fmol of the oligonucleotide primer 5' TCTTCGCTCAATAGCCC 3' complementary to positions +13 to +29 of the QCUA sequence relative to the translational initiation codon (see Fig. 2). Primer was end-labeled using polynucleotide kinase to a specific activity of 6 X lo6 dpm/pmol. Extension was carried out at 37 "C for 1 h using Moloney murine leukemia virus reverse transcrip- tase, and the products were compared with those obtained by sequenc- ing pSTA201 using the same primer.

Protein Preparation and Sequencing-ACVS protein was prepared from A . nidulans (strain G69) (17) from six 10-liter fermentation cultures grown for 32 h a t 28 "C, which are conditions that result in penicillin production. Aliquots (1 mg) of ACVS were diluted with buffer B (50 mM Tris, pH 7.5, 1 mM dithioerythritol, 0.1 mM EDTA, 10% glycerol (17)) to reduce sample conductivity to <7 millisiemens and applied to a Mono Q HR 10/10 FPLC column (Pharmacia LKB Biotechnology Inc) equilibrated a t room temperature with buffer B. Protein was eluted with a NaCl gradient (50-250 mM) made in buffer B. A total of -3.5 mg of ACVS material was isolated by this method the active fractions eluted a t between 170 and 190 mM NaCl. A final purification of ACVS was made by FPLC gel filtration of protein that had been concentrated by binding to DEAE-Fastflow (Pharmacia) and subsequently eluted with a small volume of 1 M NaCl in buffer B. Batchwise application and elution of the recovered protein through a Superose 12 column (Pharmacia) yielded homogeneously pure ACVS as analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining.

Purified ACVS was extensively dialyzed against 0.5% ammonium bicarbonate at 4 "C and lyophilized. The dry material was resus- pended (it did not redissolve) in 100% formic acid and aliquoted into -500-pmol quantities. Vaporphase pyridylethylation and CNBr cleavage of several aliquots of protein were performed (23, 24). Dried material was resolubilized in SDS gel buffer, electrophoresed, and blotted onto Immobilon-P (Millipore Corp.) (25). Amino acid se- quencing was performed directly from Immobilon-P fragments using a gas phase Applied Biosystems sequencer at the Science and Engi- neering Research Council protein sequencing facility (Department of Biochemistry, University of Aberdeen).

Subtilisin Digestion ofACVS-0.625 pg of Mono Q (FPLC)-purified ACVS was digested with 6.3 pg of subtilisin (Sigma) for 1 h a t room temperature. The reaction was terminated by addition of trichloro- acetic acid to a final concentration of lo%, and polypeptides were recovered by centrifugation. Pelleted material was solubilized in SDS gel sample buffer and neutralized by the addition of 4 M Tris prior to separation and electroblotting as described below.

Protein Analysis-Analytical electrophoresis for glycoprotein de- tection was performed with a slab gel apparatus (Hoefer Scientific Instruments) according to Laemmli (26). Proteins were separated in a 5/0.16% T/C resolving gel (where T is total gel concentration (acrylamide plus cross-linking agent, w/v) and C is cross-linker agent as a percentage of T) with 0.2% SDS in the running buffer. Protein was transferred from SDS-polyacrylamide gel electrophoresis gel onto nitrocellulose membranes (0.45-pm pore size, Schleicher & Schull) according to the method of Towbin et al. (27) (no methanol was used

in the transfer buffer) in a transfer unit (Hoefer Scientific) for 16 h at 10 "C and 2 V/cm'.

Analytical electrophoresis for molecular mass determination of native proteins was carried out using a linear polyacrylamide gradient gel system of 2-10% T a t a constant cross-linker ratio of 0.6% C in a 0.1 M Tris/HCl buffer of pH 9.0. This buffer was also used as the running buffer.

All molecular mass markers were obtained from Sigma. Glycosylation-The identification of ACVS as a glycoprotein was

carried out using ConA as an a-D-mannose- and a-D-glucose-recog- nizing reagent. ACVS preparations transferred from SDS-polyacryl- amide gels to nitrocellulose were subjected to affinity detection by sequential incubation with ConA, peroxidase, and 4-chloro-1-naph- tho1 (28). ConA-binding carbohydrate-bearing proteins develop as a purple band.

To effect removal of carbohydrate moieties, ACVS was treated with a-glucosidase (Sigma) for 15 min at room temperature (0.2 units/mg of ACVS) prior to SDS-polyacrylamide gel electrophoresis.

RESULTS

Sequence and Positive Identification of the acvA Gene Fig. 1 shows the restriction endonuclease map of the acvA

gene and the strategy adopted to determine its nucleotide sequence. The entire region was sequenced in both directions, and the nucleotide sequence along with predicted translation product is presented in Fig. 2.

Positive identification of the acvA gene and determination of the correct reading frame were obtained by comparison of the amino acid sequence predicted from the ORF with that obtained from a fragment of ACVS protein. Purified ACVS (see "Experimental Procedures") was subjected to vapor phase CNBr cleavage, and the products were electrophoresed in a 10% polyacrylamide/SDS gel; the gel was electroblotted onto Immobilon-P. A 20-kDa polypeptide was excised, and the amino-terminal sequence was determined as: Asp- Asp-Ala-Glu-Lys-Tyr-Asp-Ala-Glu-Lys-Leu-Ile-Pro- Phe-Ile. From these data, a mixed set of 23-mer oligo- nucleotides was synthesized with the sequence:

T T T A A T T 5' GACGACGCCGAGAAGTACGAcGC 3'. This mixed

probe was end-labeled and shown to hybridize to a region of pSTA201 (a plasmid capable of complementing several npeA mutants) bounded by XbaI and BamHI sites (8). DNA se- quencing of this region has shown the presence of coding potential for the amino-terminal amino acid sequence of the 20-kDa CNBr polypeptide fragment (Fig. 2, residues 547- 561), thus confirming the acuA gene as that encoding ACVS and determining the correct reading frame. This region resides in a unique ORF of 11,325 nt.

Analysis of Subtilisin Generated Fragments of ACVS Purified ACVS was digested with subtilisin and the prod-

ucts recovered by trichloroacetic acid precipitation. Polypep- tides were separated by SDS-polyacrylamide gel electropho- resis and transferred to Immobilon by electroblotting. Five bands were cut out of the blot and subjected to amino-terminal sequence analysis. All the peptides have been located within the predicted ACVS sequence and are seen to occur a t dis- persed locations throughout the protein (Fig. 2). This provides additional confirmation of the coding frame deduced from the DNA sequence of acuA.

The acuA-ipnA Intergenic Region The acvA and ipnA genes are transcribed divergently from

an intergenic sequence of 872 nt that should contain, a priori, the necessary control sequences for expression of the two genes. The complete intergenic region is presented in Fig. 2. Reading into the acuA gene, no core promoter sequences such

12648 A. nidulans ACV Synthetase

FIG. 1. Partial restriction map of the region containing the acvA gene and the sequencing strategy Z ? m E ! ? D " s o < c s .3 22: e

adopted. The solid black bar represents I P N ~ ' x m m m u m r m 1: 4 rum L y y 7 *,x 1: 5 m m

g = the approximate position of the lpnA coding region and is taken from a pre- vious report (5). kb, kilobase. " " - .

Ihb 1 - I

I T ,$ ,E I I 1 1 I I I I , I I II I I J3

""" -""" " - 4 . """A " _ " d " + " " """"""""

as TATA or CAAT motifs are obvious. This is not unusual, however, for fungal genes (29).

The 5' end of the acvA message was mapped by primer extension using RNA prepared from wild type cultures of A. nidulans grown in fermentation media, i.e., under inducing conditions (8). A major start point was found to correspond to -230 nt, whereas minor ones were located at -317, -195, and -188 nt (Fig. 3).

With regard to the ipnA gene, CAAT motifs are observed at 100 and 312 nt, whereas a TATA-like motif (TAAATAAA) is present 147 nt upstream of the ipnA ATG codon (Fig. 2).

Since acuA and ipnA expression may be regulated in a similar fashion, one might expect to observe common receptor sites for transacting regulatory proteins. Although a 53-base pair region of dyad symmetry (Fig. 2) is located within the intergenic region approximately equidistant from the ipnA and the proposed acvA initiation codons, no other extensive nucleotide sequence identities are observed between the up- stream regions of acuA and ipnA.

Analysis of the Predicted acuA Product

The unique ORF encodes a protein of 3,770 amino acid residues with a predicted molecular mass of 422,486 daltons. Since no amino-terminal sequence is available for the ACVS protein, we can only speculate on the identity of the transla- tional initiation methionine codon, but that designated is the first such codon in frame with the unique ORF. This is the first ATG codon in frame and downstream from the major transcriptional start point (see above). Additionally, sequence analysis around the AUG codon A A A m A G shows reason- able agreement with other A. nidulans translational initiation sites (29). The ORF terminates at a TAG codon. It is note- worthy that termination codons are observed in all three reading frames over the next few hundred 3' nucleotides. A potential eukaryotic cleavage and polyadenylation signal (AAATAA) is observed at nucleotide position 11,448, 134 nucleotides beyond the first stop codon.

Computer analysis of the amino acid sequence by Diagon Plot revealed three extensive regions of significant similarity.' Analysis of protein data banks also showed areas of consid- erable homology to the sequences of Bacillus brevis tyrocidine synthetase 1 (30) and gramicidin synthetase 1 (31). These sequences were aligned for maximum identity using the pro- gram BEST FIT from the University of Wisconsin sequence analysis and software package (32) and subsequently refined by eye. Fig. 4 shows the optimal alignment of the three homologous regions of ACVS and comparison with the se- quences of gramicidin synthetase 1 and tyrocidine synthetase 1. The positions of the ACVS regions, which may represent functional domains, are residues 321-910 (designated dA), residues 1413-1993 (designated dB), and residues 2494-3078 (designated dC) .

In addition to the sequence relationships shown in Fig. 4, small areas of similarity were noted with parsley 4-coumarate- CoA ligase (34) and firefly luciferase (35). The topological distribution of homologies along the polypeptide chain is

' A. P. MacCabe, unpublished results.

"" " " " " +

illustrated in Fig. 5A. The three main regions of similarity (boxes c, cys-2, and d) between ACVS, parsley 4-coumarate- CoA ligase, and firefly luciferase are shown in Fig. 5B.

Further Characterization of the ACVS Protein

Molecular Mass Determination-The acuA gene encodes a predicted protein of 422,486 Da. This is considerably larger than that expected from the first empirical determination of -230 kDa (17) by SDS gel electrophoresis. The size of the A. nidulans ACVS was, therefore, further investigated using two methods.

1) Purified ACVS was sized by FPLC gel filtration through a Superose 12 column calibrated with protein standards. The calibration curve is shown in Fig. 6, from which a size of -460 kDa can be extrapolated for ACVS. Both the UV elution profile and SDS gel electrophoresis (data not shown) indi- cated homogeneous purity of the ACVS.

2) Purified ACVS was also electrophoresed on native poly- acrylamide gradient gels (Fig. 7) and found to have a mobility corresponding to an apparent molecular mass of -420 kDa.

Both FPLC and native gel estimates of molecular mass are consistent with that determined for the translation product of the acvA gene.

Glycosylation-ACVS was identified as a glycoprotein by means of affinity blotting with ConA. 150 ng of DEAE- purified ACVS were transferred to a nitrocellulose filter from a 5% polyacrylamide SDS gel. Glycosylated protein was de- tected as a purple band upon sequential treatment of the blot with ConA, peroxidase, and 4-chloro-l-naphthol; no band developed with the glucosidase-treated ACVS sample (Fig. 8). Computer analysis of the DNA-derived amino acid sequence revealed the occurrence of 17 potential glycosylation sites (NXS or NXT) within ACVS (see Fig. 2).

DISCUSSION

Previous work based on the complementation and analysis of penicillin nonproducing A. nidulans mutants had indicated the existence of a large structural gene involved in penicillin biosynthesis located upstream of ipnA (8). The apparent size of this gene was correlated with the initial molecular mass estimate of ACVS (17) and suggested, but did not prove, a relationship between the ACVS protein and the DNA se- quences upstream of ipnA. The unequivocal identification of these sequences as the ACVS structural gene has now been achieved by matching aminoacid sequences determined from polypeptides generated from the purified protein with those predicted from the nucleotide sequence. Moreover, these data have verified the reading frame throughout the gene.

The acvA and ipnA genes are tightly linked and divergently transcribed from an intergenic region of 872 nt. As previous studies have shown that control of acvA and ipnA expression is, at least in part, at the level of transcription (8), this region could be expected to contain receptor sites for transacting elements. Analysis of the sequence has not revealed any extensive areas of similarity between the acvA and ipnA upstream regions, although a 53-base pair dyad axis of sym- metry has been located roughtly equidistant from the ipnA start site and the presumptive acvA initiation codon. The

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A. nidulans ACV Synthetase c.tTATGAATGAGGGCAAGCAGTTGAGACTGATGAAGAAGCGGTGGTAACGGAAGGATCC

CCAAGGCTTGGACTGGTTTGTGTAGGTTRATGGGGTAGGAATTGCCGGCGAAGAACAGACG -PA€

TTTCTGCGAATTTCCATCAGGGTTTATTTATTTTCCCGTCTGCGCGCAAGTCTCCAAGCA "TAAAT

AATAAGCGACCGGGTAGGACTGGCTTGGACATCGAGCCGACCACACTCGTCGCRGTA

CGAGGGGCTGTAGACAAGTATAGTCATGGCGGCCTTCGTCGGGTGACGCCTARAACCATT

ACCCTGCAGAAATTGAATCAGCGTAAGACACTGCAGCAATAGCCCCGCCAGGAATAGAGG -m GCTTGGCTCGTACCACAATTTGATGAGTAGGAATGGACTACGTCAGCCCTAAGCGAGAGA

A T A T T G G G A A A T G A T C A T A A C G A A G C T T G T T A C C C T T T T G ~ G ~ T ~ ~ ~ ~ C ~ ~ T T C ~ ~ C C ~

~~;.ABEG~SJI~Y;AC~;ETGC~~A~S~~G~T~GGACAATCTGCAGGAGACGTGAAACAT CATCTCCTGCGTACCCTATGGCACTAGTACCTAGCATGGTCTGATGCTAGGCCGTGACGG

:

TGTCGCTTGGCCGTCCCCACGGCACGGCTTGATGGTACGATGCTGCGGTACTGGATTGCC

GGGTAGCGAAGTCAGTCCCTIRRTCTT~~GAAGGCCTCTTGGCAGTGCTTCGACTGGAATC

' v .

GGCCAGGCGGAGCTGTAGTACCCCTCGTCTAGGAGGGGAGCTCGGTTTAGCACTGTGGGC

GGACTTTTCGCGGTGTCCTCACTTCTAGGCCGTCAACTGACTTCTCAAGCGATATCCCCT

M S P P G L L S CAGGCTTTTGCCCTCCGTGAAATAAGAGAATCARAATGAGCCCTCCCGGGCTATTGAGCG

E D G P G Y S G G Y A D P T V P K V N W AAGACGGCCCTGGCTACAGTGGCGGCTATGCAGACCCTACGGTGCCAAAGGTTAATTGGA

K Q S N G K S A G G N G D V D A G N G N AGCAGTCCAATGGGRGCGCCGGGGGCAATGGCGACGTTGATGCAGGCAATGGCRRCA

I D P S K S G V G V Q V C F A G G L E G TTGACCCTAGCAAATCGGGTGTTGGTGTCCAAGTGTGTTTTGCAGGAGGGCTTGAAGGTT

W K A G I S K I T E R C D L S S I A T L GGAAAGCCGGCATCAGCATAACTGAACGTTGTGATCTGAGCAGTATTGCAACAAACT

U K Y Q L A V T G F S D G P D D Y N E CGACGAAATACCAGCTTGCGGTAACCGGGTTCAGTGATGGACCGGATGACTACAATGAGT

Y S V P F P S E V L V A M E E M C L A R ACTCGGTTCCTTTTCCCTCAGAAGTACTTGTCGCGATGGAAGAAATGTGTCTTGCACGAG

D I S M R S V I Q F A V H Y V L K G F G ATATTAGTATGAGGTCTGTGATCCAGTTTGCAGTGCATTATGTGTTGAAAGGGTTCGGTG

G G S H T V A A S I D V G D D P N N I A GTGGCTCACATACTGTTGCTGCGTCGATCGATGTGGGTGACGACCCCAATAACATAGCGA

T S Y T I T P S I V C H E S R Q G Q T V CATCATACACTATTACACCCTCAATTGTCTGTCTGCCATGAGAGCAGACAAGGACAGACCGTGA

M Q E I Q S M E K L N Q L R K Q E M H P TGCAGGAGATTCAGAGTATGGRAARGTTAAACCAATTGAGGAAGCAAGAAATGCATCCGG

G E A G L S L I R M G L F D I L Y I F A GGGAGGCTGGATTAAGTCTCATCAG~TGGGGTTATTCGACATTCTGGTTATCTTCGCAG

D A N K C E G L I A G L P L A V M V C E AT(;CAAACAAGTGTGAGGGTCTAATTGCTGGCTTGCCTCTAGCAGTAATGGTGTGCGAAG

G G G R L Q V R I H F S G S L F R Q K T GAGGTGGAAGACTTCAGGTTAGAATACACTTCTCAGGGTCCCTTTTTCGACAGAAGACGT

L V D I A E A L N V L F A K A A S G G A TAGTGGATATCGCCGAAGCCCTGAACGTCTTGTTCGCTAAGGCTGCGTCGGGGGGAGCGA

T P V R D L E L L S A E Q K Q Q L E E W CGCCGGTCCGAGATCTTGAACTTCTTTCTGCAGAGC~GCAGCAGTTAGAAGAGTGGA

U D G E Y P E C K R L N H L I E E A ACAAGACGGATGGAGAGTACCCTGAATGCARAAGACTCAATCACCTTATTGAGGAGGCGA

T Q L H E D K V A I V Y K R R Q L T Y G CACAGCTGCATGAAGACAAAGTTGCCATCGTGTACAAACGTCGCCAGCTTACATACGGCG

E L N A Q A N C F A H Y L R S I G I L P AATTGAACGCGCAGGCCAACTGTTTCGCGCACTATCTGCGGTCCATCGGGATCTTACCTG

E Q L V A L F L E K S E N L I V T I L G AGCAGCTGGTGGCTTTATTTCTCGAGRAGAGCGAGCGAGAACCTTATCGTGACTATATTGGGTA

I W K S G A A Y V P I D P T Y P D E R V TCTGGAAGTCCGGCGCCGCATATGTGCCCATTGACCCAACCTACCCTGATGAACGAGTCC

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FIG. 2. Nucleotide structure of the acvA gene and deduced amino acid sequence. Numbers in the right-hand column refer to the nucleotide sequence, whereas numbers on the left represent amino acid residues. The ORF-translational end is indicated by asterisks. The intergenic region, including the ipnA translation initiation codon (i.e. cat) is shown, and nucleotide sequences referred to in the text are underlined. It should be noted that the sequence shown upstream of i p d is the nonreading strand, and consequently, putative core promoter elements in the sense strand have been included with the direction of transcription indicated by the arrowheads. The major acuA transcriptional start point is marked with a large 'I, whereas each minor one is marked with a small V. The region of dyad symmetry is marked > <. The amino acid sequences obtained from purified protein which match those predicted from the DNA sequence are boxed, and potential glycosylation motifs within the ORF are underlined.

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R F V L E D T Q A K V I I A S N H L A E GCTTTGTGCTTGAAGACACTCAGGCAAAAGTCATCATTGCGAGCAACCACCTTGCAGAGA

R L Q S E V I S D R E L S I I R L E H C GACTTCAAAGCGAGGTCATCAGCGACAGGGAGCTCTCCATTATTCGTCTAGAGCATTGCT

L S A I D Q Q P S T F P R A N L R D P S TGAGCGCCATTGATCAGCAGCCATCGACATTCCCGAGAGCCAATTTGCGCGACCCATCTC

L T S K Q L A Y V T Y T S G T T G F P K TGACCAGCAAGCAGCTTGCCTACGTTACCTATACATCGGGGACCACGGGTTTTCCGAAGG

G I L K Q H T N V V N S I T D L S A R Y GCATTCTCAAGCAACACACTAACGTGGTGAACAGCATCACTGACCTTTCAGCTCGGTATG

G V T G D H H E A I L L F S A Y V F E P GGGTGACAGGGGACCATCATGAAGCCATCCTGCTCTTTTCAGCGTATGTGTTTGAGCCCT

F V R Q M L M . A L V ' N G H L L A M ' V E TCGTGCGGCAGATGCTCATGGCACTAGTGAATGGCCATTTGCTCGCTATGGTCGATGATG

A E K Y D A E K L I P F I J R E H K I T Y CTGAGAAGTATGATGCCGRGTTGATACCATTCATTCGTGAGCACRRGATCACGTACC

L - A S V L Q E Y D F S S C P S L K TCAACGGCACTGCCTCCGTCCTGCAGGAATACGACTTCTCCTCTTGCCCATCTCTAAAGC

R L I L V G E - E S R Y L A L R R H GTTTGATCTTGGTCGGTGAGAACTTGACTGAATCTCGGTATCTGGCACTACGTAGACATT

F K N C I L N E Y G F T E S A F V T A L TCAAGAATTGCATATTGAACGAGTATGGCTTCACAGAATCAGCCTTTGTGACGGCGCTCA

N V F E P G S A R N - L G R P V R N ATGTTTTCGAACCAGGCTCGGCGCGCAATAACACGAGTCTTGGGA~CGGTGCGCAACG

V K C Y I L - L K R V P I G A T G E TCRRGTGTTATATCCTCAACAAGTCTCTCAAGCGAGTGCCTATTGGTGCCACTGGTGAAT

L H I G G L G I S K G Y L N R P D L T P TACACATTGGCGGGCTGGGTATATCCAAGGGCTACCTTAACCGTCCCGACCTTACGCCGC

Q R F I P N P F Q T D H E K E L G L N Q AACGCTTCATTCCCAACCCATTCCAAACGGACCATGAGAAGGAGCTCGGATTAAACCAGC

L M Y K T G D L A R W L P N G E I E Y L TGATGTACAAGACCGGGGATCTCGCCCGTTGGCTTCCAAACGGTGAGATCGAGTACCTCG

G R A D F Q I K L R G I R I E P G E I E GCCGCGCGGACTTCCAAATCAAGCTGCGAGGGATCCGTATCGAGCCCGGCGAGATAGAGT

S T L A G Y P G V R T S L V V S K R L R CCACTCTGGCGGGTTACCCTGGGGTACGAACCAGCCAGCCTAGTCGTCTCT~GGTTGCGGC

H G E K E T T N E H L V G Y Y V G D - ATGGCGARAAGGAGACTACCAACGAGCATCTGGTAGGCTATTATGTGGGCGATAATACCT

S V S E T A L L Q F L E L K L P R Y M I CTGTCTCTGAAACGGCTCTCTTGCRRTTTCTGGAGCTGAAGCTGCCCCGATACATGATTC

P T R L V R V S Q I P V T V N G K A D L CGACACGACTTGTGCGCGTGTCTCAAATCCCAGTGACTGTTAATGGAAAGGCAGACCTCC

R A L P S V D L I Q P K V S S C E L T D GTGCCCTACCTTCTGTCGACCTTATTCAACCCAAAGTGTCCTCTTGCGAGCTCACGGATG

E V E I A L G K I W A D V L G A H H L S AGGTGGAAATAGCTTTGGGGARGATRTGGGCRGATGGGCAGATGTTCTCGGAGCCCATCACCTGTCGA

I S R K D N F F R L G G H S I T C I Q L TATCCCGTAAAGACAACTTCTTTCGTCTTGGAGGGCACAGCATCACATGCATCCAGCTCA

TCGCACGTATTCGCCAGCAGCTTGGTGTAATTATTTCCATTGAGGACGTTTTCTCATCCC I A R I R Q Q L * G V I I S I E D V F S S

R T L E R M A E L L R S K E S L P D GGACACTGGAGCGTATGGCTGAGCTTCTGCGRRGCAAAGAGTCCAACGGAACTCCGGATG

E R A R P Q L K T V A G E V A I N A N V Y AGAGGGCTAGGCCTCRRCTACCGTGGCGGGAGAAGTTGCAAATGCTAATGTCTATC

L A N S L Q Q G F V Y Q F L K N M G R S TTGCTAACAGTCTCCAGCAAGGCTTCGTTTATCAGTTCCTG~TATGGGCCGATCAG

E R Y V M Q S V L R Y I D V N I N P D L F AGGCTTATGTGATGCAATCCGTGCTGCGATACGATGTCAATATCAATCCTGATCTATTTA

K K A W K Q V Q H M L P T L R L R F Q W AGCCTGGAAGCAGGTACAACACATGCTTCCAACACTGAGGCTCCGATTTCAATGGG

G Q D V L Q V I D E D Q P L N W W F L H GACAGGATGTTTTGCAGGTGATTGACGAGGACCAGCCGCTGAACTGGTGGTTCTTACACC

L A D D S A L P E E Q K L L E L Q R R D TTGCCGACGATTCAGCCCTGCCCGAGGAGCAGAAACTACTAGAGTTACAGCGCAGGGACC

L A E P Y D L A A G S L F R I Y L I E H TGGCTGAGCCATACGACCTAGCAGCCGGAAGCCTGTTCCGCATTTATCTGATCGAGCATA

S S T R F S C L F S C H H A I L D G W S GCTCAACTCGGTTTTCGTGCTTGTTCAGCTGTCATCACGCAATCCTTGATGGATGGAGCC

L P L L F R K T H G T Y L H L L H G H S TGCCGCTTCTTTTCAGGAAGACTCATGGAACTTATCTGCATCTCCTGCACGGACATTCTC

L R T L E D P Y R Q S Q Q Y L Q D H R E TCAGGACTCTGGARGACCCTTACAGGCAGTCTCAGCAGTATCTCCAAGATCATCGCGAAG

D H L R Y W A G I V N Q I E E R C D M N RTCATCTCAGGTACTGGGCTGGTATCGTGAATCAGATTGAAGAGCGTTGTGACATGAACG

A L L N E R S R Y K I Q L A D Y D K V E CTTTGCTGAACGAACGCAGTCGGTACAAGATTCAACTGGCGGACTATGACAAAGTGGAGG

FIG. 2"Continued

12649

1285

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1585

1 6 4 5

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1945

2005

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A. nidulans ACV Synthetase 1169

1189

1209

1229

1249

1269

1289

1309

1329

1349

1369

1389

1409

1429

1449

1469

1489

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1929

D Q Q Q L T L T V P D A S W L S K L R Q ATCAACAACAATTAACTTTAACAGTCCCTGATGCTTCCTGGCTAAGCAAATTGCGCCAAA

T C S A Q G I T L H S I L Q F V W H A V CATGCTCTGCGCAAGGCATTACATTGCACTCTATTCTGCAGTTTGTTTGGCACGCGGTAT

L H A Y G G G T H T V T G T T I S G R N TGCATGCTTACGGTGGCGGTACTCATACTGTCACTGGCACTACTATCTCAGGGAGGAACC

L P V S G I E R S V G L Y I N T L P L V TGCCTGTGAGTGGGATCGAACGATCTGTGGGTCTCTACATAAATACGCTCCCACTGGTAA

I N Q L A Y K - V L E A I R D V Q A TTAATCAGTTGGCCTATAAGAATAARACCGTCTTGGAGGCTATCCGTGATGTGCAGGCCA

I V N G M N S R G N V E L G R L Q K N E TTGTAAATGGCATGAACAGCCGGGGAAATGTGGAACTTGGCCGTCTACAGAAAAACGAGC

L K H G L F D S , L F V L E N Y P I L D K TGAAGCATGGGTTATTTGACTCGCTATTTGTGCTGGAGAATTATCCAATACTGGACAAGT

S E E M R Q K S E L K Y T I E G N I E K CCGAGGAGATGCGGCAGAAGAGTGAATTGAAGTATACCATCGAAGGCAATATTGAARRGC

L D Y P L A V I A R E V D L T G G F T F TCGACTATCCCCTTGCTGTTATCGCGCGCGAGGTCGACCTAACTGGGGGATTCACCTTCA

T I C Y A R E L F D E I V I S E L L Q M CCATCTGCTACGCTCGAGAGCTTTTCGATGAGATTGTTATATCTGAGTTGCTCCAAATGG

V R D T L L Q V A K H L D D P V R S L E TCCGGGACACGCTCCTGCAAGTCGCGAAGCATTTAGATGACCCCGTCCGCAGCCTAGAGT

Y L S S A Q M A Q L D A W U D A E F ATCTGTCATCAGCGCAAATGGCTCAACTTGACGCATGGAATGCGACAGACGCGG~TTCC

P D T T L H A M F E K E A A Q K P D K V CCGACACCACCCTACACGCGATGTTCGAAAAAGAAGCGGCCCAGRAACCAGACAAGGTCG

A V V Y E Q R S L T Y R Q L N E R A N R CGGTGGTCTATGAGCAACGCAGCTTGACGTATCGTCAGCT~TGAGCGGGCGAACCGTA

M A H Q L K S D I S P K P N S I I A L V T G G C G C A C C A G C T C A A A T C T G A T A T C A G C C C A A A G C C G A R

V D K S E H M I A T I L A V W K T G G A TGGATAAGAGTGAGCATATGATAGCTACCATTCTGGCTGTGTGGAAGACTGGCGGTGCCT

Y V P I D P E Y P D D R I R Y I L E D T ATGTACCGATCGACCCTGAGTACCCCGACGACCGTATCCGCTATATCCTAGAAGACACCA

S A I A V I S D A C Y L S R I Q E L A G GCGCCATTGCCGTGATTTCAGACGCGTGTTACCTCTCACGAATCCAAGAATTAGCGGGAG

E S V R L Y R S D I S T Q T D G - V AGAGTGTCCGTCTGTATCGGTCTGACATCTCTACTCAGACTGACGGTAACTGGAGTGTGT

S N P A P S S T S T D L A Y I I Y T S G CGAATCCTGCACCGTCCAGTACGAGCACGGATCTTGCATATATTATCTACACTTCGGGAA

T T G K P K G V M V E H H G V V N L Q I CAACTGGGAAGCCAAAGGGCGTCATGGTGGAGCACCACGGAGTGGTAAATCTGCAGATAT

S L S K T F G L R D T D D E V I L S F S CGCTGTCTAARRCCTTCGGGCTGCGCGATACTGATGACGAGGTAATCCTCTCATTCTCCA

N Y V F D H F V E Q M T D A I L N G Q T ACTACGTCTTTGACCATTTCGTGGAACAGATGACGGATGCCATTCTCAACGGCCRAACAT

L V M L N D A M R S D K E R L Y Q Y I E TAGTTATGCTCAACGATGCAATGCGCAGTGACAAAGAGCGCCTCTACCAATATATCGAAA

T N R V T Y L S G T P S V I S M Y E F S CTAATAGGGTAACATACCTGTCTGGAACCCCATCCGTTATTTCCATGTATGAGTTCAGTC

R F K D H L R R V D C V G E A F S Q P V GATTTAAAGACCACCTACGCCGTGTCGACTGCGTTGGAGAAGCTTTTAGCCAGCCCGTCT

F D Q I R D T F Q G L I I N G Y G P T E TTGATCAAATCCGTGACACTTTCCAAGGGCTGATTATCAACGGCTACGGTCCAACAGAGA

TCTCCATCACGACACACAAGCGGCTGTACCCTTTCCCTGAGCGGCGCACAGATAAGAGCA I S I T T H K R L Y P F P E R R T D K S

I G Q Q I G N S T S Y V L N A D M K R V TCGGCCAGCAGATTGGCAACAGTACGAGCTACGTGCTGAATGCAGACATGAAACGCGTTC

P I G A V G E L Y L G G E G V A R G Y H CAATTGGGGCTGTAGGTGAGCTCTATCTGGGTGGTGAAGGCGTCGCGCGAGGATATCATA

N R P E V T A E R F L R N P F Q T D S E ACCGACCGGAAGTGACTGCTGAGCGATTTTTACGCAATCCGTTCCAAACAGACAGTGAAC

R Q N G R N S R L Y R T G D L V R W I P GGCAARRTGGGCGCAACAGCCGCTTGTACAGGACCGGTGACTTGGTACGCTGGATCCCAG

G S N G E I E Y L G R N D F Q V K I R G GCAGTAACGGTGAAATTGAATATTTGGGACGCAATGACTTCCAGGTCAAGATTCGCGGGC

L R I E L G E I E A V M S S H P D I K Q TCCGTATCGAATTGGGGGAGATTGAGGCTGTCATGTCCTCACATCCTGACATTAAACAGT

S V V I A K S G K E G D Q K F L V G Y F CTGTTGTAATTGCAAAGAGTGGCAAGGAAGGAGACCAGAAGTTCCTTGTTGGTTACTTCG

V A S S P L S P G A I R R F M Q S R L P TGGCTAGCTCGCCATTGTCTCCGGGTGCAATCCGGCGCTTTATGCAATCCCGGCTTCCTG

G Y M I P S S F I P I S S L P V T P S G GCTATATGATACCTTCAAGTTTCA~TCCTATCAGTTCTCTCCCAGTGACTCCCAGTGGAA

K L D T K A L P T A E E K G A M N V L E AGCTGGATACRAAGGCCTTACCTACAGCAGAGGAGAAAGGCGCAATGAACGTGCTGGCTC

P R N E I E S I L C G I S A G L ~ L D I S CACGTAATGAAATCGAGAGCATCCTGTGCGGTATCTCGGCAGGGTTGTTAGATATATCCG

FIG. 2-Continued

3565

3625

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4105

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5 0 0 5

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1949

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1989

2009

2029

2049

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2709

A Q T I G S D S D F F T L G G D S L K S CCCAAACRATTGGCAGCGACAGCGATTTTTTCACCCTCGGAGGCGATAGTTTGAAGAGTA

T K L S F K I H E V F G R T I S V S A L CAAAGCTCTCATTCAAGATTCACGAGGTATTTGGCffiCACAATCTCCGTCAGCGCTCTGT

F R H R T I E S L A H L I M N N V G D I TCCGTCACCGAACCATCGAGAGTCTGGCACACCTRRTTATGAACAATGTTGGAGACATAC

Q E I T P V D Y D N R R K I R V S P A Q AGGAGATCACGCCTGTGGATTATGATGATAACAGACGC~TAGCCGTATCTCCCGCTCAAG

E R L L F I H E L E G G G N A Y N I D A AGCGCCTTCTATTCATTCACGAGCTTGAAGGTGGAGGCAATGCATATAATATCGATGCTG

A F E L P P Y I D Q S R V E E A L Y T I CCTTTGAGCTACCTCCATACATTGATCAATCTCGAGTCGAAGAGGCATTATATACCATTC

L S R H E A L R T F L L R D Q A T G T F TTTCAAGACACGAAGCCTTACGAACATTTCTGCTGCGGGACCAGGCAACTGGCACGTTCT

Y Q K I L T T D E A K C M L I I E K S A ACCAARAGATATTGACTACCGATGAGGCCAAGTGCATGTTGATCATTGAGAARRGTGCAG

V S T I D Q I D S I V G R L S Q H I F R TGAGCACCATTGATCAAATTGATTCCRTWTCGGACGCCTATCGCAGCACATTTTCCGTC

L D S E L P W L A H I V T H K T G N L Y TCGATTCTGAGCTTCCCTGGTTGGCGCATATTGTCACGCAC~CGGGCAATCTTTATC

L T L S F H H T C F D A W S L K I F E R TGACCCTGTCCTTCCATCACACTTGCTTCGATGCATGGTCATTGAAGATCTTCGAGCGGG

E L R V F C A S K Q K G G N M P I L P M AGCTCCGCGTTTTTTGCGCGTCAAAGCAAAAAGGCGGCAACATGCCAATCCTACCAATGC

P Q V Q Y K E Y A E H H R R R L G K N Q CTCAAGTCCAGTACAAGGAGTATGCCGAGCACCATCGTCGACGACTAGGTAAGAATCAGA

I Q K L S D F W L Q R L D G L E P L Q L TTCAAAAATTATCCGACTTTTGGCTGCAAAGACTAGACGGCCTGGAGCCCCTACAGCTCC

L P D Y P R P A Q F N Y D G G D L S V I TACCGGATTATCCGCGGCCTGCCCAATTC~CTACGATGGAGGTGACCTCTCCGTCATTC

L D G V V L E T L R G I A K D H G V T L TGGACGGTGTGGTTCTGGACCCTCAGGGGCATTGCAARRGACCACGGAGTAACTCTGT

Y A V L L A V V C L M L S T Y T H Q V D ACGCAGTGCTTCTCGCTGTTTACTGCCTGATGCTTTCGACATATACACACCAGGTAGATA

I A V G V P I S H R T H P L F Q S I V G TCGCTGTGGGAGTCCCCATCAGTCACCGAACCCACCCCCTGTTCCAGTCTATTGTCGGAT

F F V N M V V V R V D V K D F A V H D L TCTTCGTCAATATGGTAGTTGTGAGGGTCGACGTGAAGGACTTTGCCGTTCACGATCTCA

I R R V M K P L V D A Q L H Q D M P F Q TTCGAAGGGTAATGAAACCGCTTGTTGATGCCCAGTTACATCAGGACATGCCATTCCAAG

D V T K L L R V D N D A S R H P L V Q T ACGTGACTIU\ACTGCTGCGGGTGGATAACGACGCCAGCCGACATCCCCTAGTTCAGACTG

V F N F E S D M D K E F E T T P S I Q D TGTTCAACTTTGAAAGTGACATGGACAAAGAATTCGAGACGACACCTTCAATCC~GACA

T A T I A P Y Q S V Q R I K S V A K F D CTGCCACAATCGCACCATACCAGTCCGTTCAGAGGATAAAGTCGGTTGCGRAATTTGATC

L U A T E S G S A I L K I N F N Y A T TGAACGCGACAGCTACAGAGTCGGGCTCAGCCTTRAAGATTAACTTTAACTATGCCACCA

S L F R K E T I Q G F L E T Y R H L L L GCCTGTTCCGGRAAGAAACGATCCAGGGCTTCTTAGAGACATACAGGCATCTCCTGTTAC

Q L S Y L G S Q G L K E D T K L L L V R AGCTCTCTTATCTGGGGTCCCAGGGACTTAAAGAAGATACAAAGCTACTGTTGGTCCGCC

P E E M S G P H L P L A G L S N G A E T CTGAGGAGATGAGTGGTCCGCATCTGCCATTAGCAGGATTATCCAATGGTGCGGAAACCC

L E R I S L S R A F E F E A F R V P D R TAGAAGCTATATCACTCAGTAGAGCATTCGAGTTTGAAGCTTTCAGGGTACCGGATAGAG

A A V V Q G D K S L S Y T E L N K R A N CTGCCGTCGTACAGGGAGATAAATCACTCA~CTATACCGAGCTCAATAAACGGGCAAACC

Q L A R Y I Q S V A H L R P D D K V L L AGCTAGCCCU~TACATACAATCCGTGGCACACCTTAGGCCGGACGACAAGGTGCTCCTCA

I L D K S I D M I I C I L A I W K T G S TTCTGGATAAGAGCATCGACATGATTATTTGCATCCTCGCAATCTGGAARRCCGGTAGCG

A Y V P L D P S Y P K E R V Q C I S E V CATATGTGCCTTTGGATCCATCATATCCCAAGGAGCGTGTCCAGTGCATTTCGGAGGTAG

V Q A K I L I T E S R Y R S A W G S Q T TTCAAGCRAAGATTCTGATTACAGAGTCACGGTACGCCTCTGCATGGGGAAGCCAGACGT

S T I L A I D S P K V S N M V N N Q A T CAACAATACTTGCAATTGACTCGCCCAAGGTCTCGAATATGGTCAATAATCAGGCAACT~

H N L P N I A G I K N L A Y I I F T S G ATAACTTGCCCAACATTGCGGGAAT~TCTGGCATATATAATTTTCACATCTGGCA

T S G K P K G V L V E Q G G V L H L R D CCTCCGGCAAGCCAAAGGGTGTTCTGGTCGAACAAGGTGGAGTTCTTCACTTGCGTGATG

A L R K R Y F G I E C N E Y H A V L F L CGCTTAGGAAGCGGTACTTTGGCATTGAATGCAATGAATACCATGCTGTGCTCTTCCTAT

S N Y V F D F S I E Q L V L S I M S G H CCAATTACGTGTTTGATTTCTCTATCGAGCAGTTGGTCTTATCAATTATGAGCGGCCACA

K L I I P E G E F V A D D E F Y I T A N

FIG. 2-Continued

5905

5965

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6265

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6385

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1645

1 1 0 5

7765

7825

7885

7945

8005

8065

8125

A. nidulans ACV Synthetase 12651 AGTTGATCATCCCGGAAGGAGAATTCGTTGCGGATGATGAATTCTACATAACAGCCAACG

G Q R L S Y L S G T P S L L Q Q I D L A GTCAACGCCTCTCATATTTGAGCGGTACACCATCCCTGTTGCAGCARRTTGACCTAGCAC

R L N H L Q V V T A A G E Q L H A A Q F GCCTCAATCATCTACAGGTCGTRRCTGCAGCTGGTGAGCAACTCCATGCTGCGCAGTTTA

N K L R S G F R G P I Y N A Y G I T E T ATRRGTTGCGCTCCGGATTCCGCGGCCCGATCTACAACGCATATGGAATTACGGAGACCA

8185 3469

3489

3509

3529

3549

A D R D Q S S F A V D I T A S C V N G A CGGACAGAGACCARRGCTCGTTCGCGGTTGATATCACCGCCAGCTGTGTARRTGGTGCCC 10465

2129

2749

2169

2789

2809

2829

2849

2869

2889

2909

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2989

3009

3029

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8905

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9145

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9265

9325

L S V E M N S A W S L E K S M R F I S R TGTCAGTCGARRTGRATAGTGCCTGGAGCCTTGAAAAAACATGCGATTCATATCCAGGA

I E E V L N M I L S G T L A Q Q A T P V TTGAGGAAGTATTGAATATGATTCTTAGCGGGACCCTAGCTCAGCAGGCGACTCCAGTGC

10525

10585

L T P Q V F N E E M Y T P Y F E F S K T TTACGCCACAGGTATTCAACGAGGAGATGTACACACCATATTTTGAATTTTCC~CCC

P R R G P I L F L L P P G E G G A E S Y CACGACGCGGACCGATCTTGTTCCTATTGCCGCCAGGGGAGGGAGGGGCAGARRGCTACT

10645

10705

T V Y N I V S E F S A Q S Q F E N A L R CGGTATACAACATAGTCAGCGAGTTCAGTGCGCAATCCCAATTCG~TGCTCTGCGAG

E L L P G T R A Y L L N H A T Q P V P M AGCTGCTACCAGGCACTAGGGCATATCTTCTTAACCACGCCACTCAGCCAGTTCCTATGA 3569

3589

3609

3629

3649

3669

3689

3109

3129

3149

3769

F N N I V K H L P T T N N V V F N N Y Y TTRRCAATATCGTCRRGCACTTGCCCACGACTAATATGGTCGTCTTTAACAATTACTACC 10765

10825

N A V G E L Y L A G D C V A R G Y L N Q ACGCAGTCGGAGAGCTGTATCTCGCTGGTGATTGTGTGGCCCGTGGCTATCTCRACCAGC

P V L T G D R F I Q N P F Q T E Q D I A C T G T T C T R A C A G G T G A C C G T T T T A T C C A G M T C C A T T C C A G A T A T T G C T T

C G S Y P R L Y R T G D L F R C R L D R GCGGAAGCTATCCTCGGCTCTATAGAACTGGCGACCTGTTTCGATGCCGGCTTGACCGTC

L H S K S L N T F E K L A E M Y L G H I TTCACTCCAAGAGTCTGAACACGTTTGRRAAGCTAGCTGAGATGTATTTGGGGCACATCC

R Q I Q P D G P Y H F I G W S F G G T I GTCAGATCCAGCCAGACGGGCCTTACCATTTCATCGGATGGAGTTTTGGAGGAACAATCG 10885

A M E T S R Q L V G L G S T I G L L G I CGATGGARRTATCGCGACAGCTCGTGGGGCTAGGTTCRACGATTGGTCTTTTAGGTATCA 10945

11005

Q H Q P Y L E Y L G R A D L Q V K I R G AGCACCAGCCATATCTAGAATATCTTGGRAGAGCTGATCTCCAGGTCAAGATRAGAGGAT

Y R I E P S E V Q N V L A S C P G V R E ACCGTATTGAGCCGTCAGAAGTTCAGMCGTGCTTGCTTCCTGTCCTGGCGTTCGAGAAT

C A V V A K Y E N T D A Y S R I A K F L GTGCAGTAGTGGCCAAGTATGAGAACACCGATGCTTACTCCAGGATAGCCARRTTCCTGG

I D T Y F N V P G A T R A I G L G D T E TTGACACGTATTTCAACGTGCCTGGAGCAACGCGGGCRRTTGGCCTCGGTGATACTGAGG

V L D P I H H I S Q P E P A D F Q C L P TCTTGGATCCCATTCATCATATATCCCAACCAGAACCAGCCGATTTCCAGTGCCTCCCAG 11065

A S T D Y I I L F K A T R V N D K F Q S CCAGCACAGACTACATCATTTTATTCAAAGCTACTAGGGTGAACGACAAGTTTCAGTCTG 11125

11185

V G Y Y T P D T E T V S D S S I L A H M TCGGATATTATACCCCTGACACCGAGACGGTCTCCGATTCAAGTATCCTCGCCCACATGA

K S K L P A Y M V P K Y L C R L E G G L ARRGCAAGCTTCCCGCATATATGGTCCCTARRTATCTATGCCGTCTAGAAGGTGGACTTC

P V T I N G K L D V R K L P D I G N P Q CAGTGACAATCAACGGGARRCTTGACGTTCG~GCTGCCTGATATCGGCAACCCTCRAC

E N Q R R L Y E Y Y D K T L L N D L D W RRAACCAGAGGCGTCTGTACGAGTACTACGACRRAACATTGCTTAATGATCTCGACTGGT

L L P G A S N I H L V R L E E D T H F S T A C T C C C T G G T G C T T C A R R C A T T C A T C T A G T C C T T G A T T C T C C T 11245

W A T N P R Q I A H V C S T I E K F L A GGGCGACCAATCCACGCCARRTCGCCCACGTTTGTTCAACAATCGAGARRTTTCTCGCCA 11305

H Q I S Y N P P R D V L E A D L C R L W ATCARRTATCGTACAACCCCCCAAGGGATGTCCTGGAGGCCGACTTGTGTAGATTATGGG

A S A L G T E R C G I D D D L F R L G G CATCAGCACTAGGAACAGAGCGATGCGGTATTGATGATGATCTGTTTAGGTTAGGCGGAG

R Y . ' GATATTAGTRRAGTGATGCCAACAACGTATGCATRRTGAGGTGGATGTAATCCAGCAAGG

TCAGGTTCGGGGCAGGATTTTGTGCTGTAACTCACTTTACGTGGATATAT~TCTTTAGAG

11365

11425

11485

11502

D S I T A L H L A A Q I H H Q I G R K V ACAGTATTACTGCTTTGCATCTCGCAGCCC~TCCACCACCAGATCGGCCGARRGGTCA

T V R D I F D H P T I R G I H D N V M V CTGTTCGAGATATTTTCGACCACCCTACCATTCGTGGTATTCATGACAACGTTATGGTGA

K L V P H N V P Q F Q A E I Q Q T V L G D AACTCGTTCCACACAATGTTCCTCRATTCCRAGCAGAGCAGC~CAGTACTCGGTGATG

AGACAATAATCRRTCATACTTCAAATAATGARRCGAACCTGCTCATTGCWCATGCCT

AAGCAGCACAGCAACCC

FIG. 2-Continued

3089

3109 A P L L P I Q I W F l L S K S L Q H P S H CGCCTCTGCTACCGATCCATTTGGTTCTTATCAAAATC~TCGCTACAGCACCCRAGCCATT 9385

3149

3169

3189

3209

3229

3249

3269

3289

3309

3329

V A E L Q L Y H D A F R M R L R Q I D G TCGCCGAATTGCAGCTGTATCATGACGCCTTCAGAATGCGGTTGAffiCARRTAGATGGAA

R T V Q C F A D D I S P V Q L R V L N V GGACGGTGCAATGCTTCGCAGATGACATTTCTCCAGTACAGCTCCGAGTGTTGAACGTCA

K D V D G S A A I D Q Q L Q K Y Q S D F AGGATGTCGACGGAAGCGCGGCTATTGACCAGCRRCTCCAGARRTATCAGTCTGACTTCG

D L E K G P I C A A A Y L H G Y E D R S ACCTTGAGARRGGCCCAATCTGTGCTGCTGCCTACCTCCATGGCTACGAGGATCGATCTG

A R V W F S V H H I I I D I V S W Q I L CACGAGTCTGGTTTTCTGTCCACCACATCATCATTGATATAGTTAGCTGGCAGATTCTTG

A R D L Q I L Y E G G T L G R K S S S V CGCGCGACCTACRRRTCCTGTACGAGGGTGGAACTCTCGGTCGTAAGAGTAGCAGCGTCA

R Q W A E A L Q S Y Q G S A S E R A Y W GACRATGGGCAGAGGCACTACAGAGCTACCAGGGGTCGGCATCGGAGAGGGCCTACTGGG

E G L L A Q T A A N I S A L P P V T G T AAGGACTTCTTGCTCARRCGGCTGCCRACATATCCGCTTTGCCCCCAGTGACCGGGACCC

R T R L A R T W S D D R T V I L L N E A GTACCCGGTTGGCTCGAACTTGGAGTGACGACAGGACffiTCATTCTCCTGAATGAAGCTT

S N Q - I Q D L L L A A V G L A L Q CTAATCAGAATGCATCTATACRRGACCTCTTACTCGCCGCTGTTGGATTGGCACTTCRRC

Q V T P G S P S M I T L E G H G R E E I AGGTCACCCCGGGTAGCCCGAGTATGATTACTCTCGAGGGCCATffiGCGTGAGGARRTTG

V D P T L D L S R T L G W F T S M Y P F TTGACCCGACATTAGACCTCAGCCGTACCTTGGGTTGGTTCACCAGCATGTATCCCTTCG

E I P P L N V E T L S Q G I A S L R E C AGATCCCTCCCCTGAATGTTGARRCCCTTAGCCAGGGCATAGCCAGCTTGCGAGAATGCC

L R Q V P A R G I G F G S L Y G Y C K H T T A C ~ ~ A O C I T C C C T r C L r r ~ r ~ ~ ~ T r ~ ~ ~ T T T ~ ~ ~ T r ~ r T r ~ ~ r ~ ~ ~ ~ ~ ~ ~ ~ r ~ ~ ~ r ~ r ~

Q M P Q V T F N Y L G Q L T S K O S I T

9505

9565

9625

9685

9145

9805

9865

9925

9985

10045 FIG. 3. Pr imer ex tens ion of the acvA transcript . End-labeled

Drimer was hvbridized overnight at 60 "C to 100 pg of yeast mRNA 3349

3369

3389

3409

3429

10105

10165

10225

1 0 7 0 c

ilune I ) and io0 pg of total KNA from mycelium grown in fermen- tation medium (lune 2) . Large 4, the major transcriptional point; small 4, the minor points that are visible on the autoradiograph. Following extension with reverse transcriptase, reactions were ethanol-precipitated and RNase A-treated for 1 h a t 37 "C, and the total product was analyzed on a 6% polyacrylamide/urea sequencing gel against a pSTA201-sequencing ladder generated using the same primer. AutOraUlOffraphy was for 14 clays wlth KOUak XAK. tllrn.

ARRTGCCTCAGGTTACGTTCAACTACCTGGGCCAGCTGACAAGCAAGCAATCGATAACTG 10345

3449 D Q W A L A V G D G E M Q Y G L T T S P significance of this region is not currently known. Availability

ATCAGTGGGCCCTCGCTGTTGGTGACGGAGAGATGCAATATGGGCTTACAACRRGTCCTG 10405 of the intergenic sequence should facilitate rigorous analyses by functional tests.

The acuA gene appears to be intron-less, a feature that is FIG. 2"Continued

12652

dA 321 dB 1413 dC 2494

GS1 41 TYl 29

dA 379 dB 1412

GS1 99 dC 2553

TY1 87

dA 439 dB 1525 dC 2613

GS1 153 TYl 141

dB 1584 dA 498

GS1 208 dC 2664

TY1 196

dA 557 dB 1643 dC 2723

GS1 265 TY1 252

dA 616

dC 2182 dB 1103

GS1 322 TY1 309

dA 675 dB 1162 dC 2840

GS1 381 TY1 369

dA 129 dB 1818

GS1 427 dC 2898

TY1 415

dA 789 dB 1873 dC 2955

GS1 481 TYl 469

dA 841 dB 1930

GS1 539 dC 3017

TY1 528

dA 906 dB 1989 dC 3074

GS1 596 TY1 585

A. nidulans ACV Synthetase

VNSITDLSARY.GVTGDHHEAILLFSAYVFEPFVRQMLMALVNGHLLAMVDDAEKYDAEK **LQIS'*KTF.'LRDTDD'V'*S**N*t*DH**E**TD*IL**QT*V*LN**MRS~K~R LHLRDA*RK*'F*IECNEYH*V*FL*N"*rDFSIE'LVLSI~S**K~.IIPEG'FVADDE S*LKVFFENSL.N**EKDR..*GQ*ASIS*DAS*WE*F***LT~AS'YIILKDTIN'FV+ AICNPFSKI*L.ASPSKTG..SG.FLPACRSTHPFGKCSW'CCIRVHPSKQTIH*FA?a

SVSETALLQFLELKLPRYMIPTRLVRV.SQIPVTVNGKAOLRALPSVDLI.QPKVSSCEL PL'PG*IR*MQSR**G**k*SSFIPI.*SL***PS*rL*TK+**TAE.E.KGAMNVLAP T*~DSSI*AHMKS***A*'V*KY*C'LEGGII'**I*'.L~V*K*~DIGNP.'HQI'YNPP HIPLEQ*R**SSEE**T****SYFIQL.DKM*L*Sr+,I.RKQ*.EP*~T.FGMRVDY.A ERTPAQ'RDYAAQ'"A+'L'SYF'KL.DKM'L'P'D*I*RK**~EP**TANPSaARYBP

FSSRT *RH**

LKYP' 'DHP*

LNYP*

378 1471 2552

98 86

438

2612 1524

152 140

1583 497

2663 207 195

556 1642 2722 264 251

615

2781 1702

321 308

674

2839 1761

380 368

728 1817 2897

414 426

788

2954 1872

480 468

1929 846

3016 538 527

905 1988 3013 595 584

FIG. 4. Alignment of homologous amino acid regions: ACVS domains A, B, and C, gramicidin S synthetase 1 (GSI), and tyrocidine synthetase 1 (TYI ) . The locations of these regions are indicated. Relative to domain A ( d A ) , identical residues are marked with an asterisk; small gaps have been introduced to give optimal alignment. Boxes a-W contain the stretches of greatest sequence similarity between all five polypeptides. Boxes cys-I, cys-2, and cys-3 represent regions of similarity between TY1 and GS1 associated with conserved cysteine residues. These cysteines however, are missing in ACVS. The TGD motif, found in ATPases (33), is present in all five polypeptides analyzed.

rather unusual for filamentous fungal genes, as most contain small introns (50-200 nucleotides) (29). With regard to gene structure and expression, the acuA gene exhibits little codon preference (data not shown), a feature generally associated with poorly expressed genes (29).

It has been shown that ACVS contains three homologous regions of about 585 amino acids separated by unrelated regions. From known data on the organization of gramicidin synthetase 2 (19) we propose that the homologous regions of approximately 600 amino acids catalyze the activation of amino adipate, cysteine, and valine, in that order. Areas of high amino acid sequence similarity have been found at cor- responding distances in the three homologous ACVS regions, gramicidin synthetase 1, tyrocidine synthetase 1, parsley 4- coumarate-CoA ligase, and firefly luciferase (Figs. 4 and 5B). Since all of these enzymes form adenylates, these common "motifs" may be significant for this reaction. As the thiotem-

A.

B.

C .

D.

LUC - S" I

0 100 -

box c : ACVS dA 472 4CL 181 LUC 198

box cys-2: ACVS dA 667 4CL 460 LUC 412

box d: ACVS dA 729 4CL 508 LUC 520

TY 1 GS1

553

ACVS dA 812 564

ACVS dB 1955 ACVS dC 3040

TY2 GS2

45

ACVS 1286 45

500 1000 amino-acid residue

KQLAYVTYTSGTTGFPKGILKQHTN DDW*LP*S*****L***VMLTHKG *TI*LIMNS**S**L'**VALPHRT

GELHIGGLGISKGYLNRPDLTPQRFIP **IC*R'DQ*M*****D*ES'RTTIDE ***CVR*PM*MS**V*N*EA*NALIDK

GRADFQIKLRGIRIEPGEIESTLAGYP D*LKEI**YK*FQVA*A*L*AL*LTH* D*LKSL**YK*YQVA*A*L'*I*LQH*

.DNFYSLGGDSIQAIQWARLHSYQLKLET

K***FR***H**TC**LI**IRQQLGVIIS ****AI*****K****A************

DSD*FT*****LKSTKLSFKI*EVFGRTIS D*DLFR******T*LHLA*QI*HQIGRKV*

496 205 212

486 693

498

755 534 546

581 592 901

1984 3069

74 74

1314

FIG. 5. Amino acid sequence relationships between ACVS and other polypeptides. A , topological distribution of regions of similarity between tyrocidine synthetase 1 (TYI) , gramicidin S syn- thetase 1 (GSI), luciferase (LUC), 4-coumarate-CoA ligase (4CL), and ACVS domains dA, dB, and dC. The respective regions were initially defined as areas of high similarity between TY1 and GS1. Refer to the legend to Fig. 4 for the amino acid sequence within boxes a-W. The consensus sequences derived from TY1 and GS1 in the boxes located outside the ACVS domains are: box e. 667- KI.EHHDA.RM-677; box /, 750-AIHHLVVDGISWRILF-765; box g, 906-ARTVGWFTSQYPV-918; and box h, 974-FNYLGGFD.D..TE LFTRSPY-995 (numbering is based on gramicidin synthetase 1). The dots indicate positionally conserved cysteine residues, termed cys-I to cys-4. The adjacent high similarity areas cys-2 and TGD have been fused to form a single box in the ACVS domains, since there is significant sequence conservation in the intervening regions. B, align- ment of main areas of sequence similarity between the ACVS domain, represented by dA, 4CL, and LUC. C, alignment of sequences con- taining the conserved LGGDS/LGGHS motifs (W box region). D, alignment of sequences of ACVS, tyrocidine synthetase 2 (TY2) and gramicidin synthetase 2 (GS2) containing the DSL-related motif. Relative to the upper sequence of each alignment detailed, identical residues are marked with an asterisk.

plate mechanism (thought to involve the presentation of activated amino acids and peptide intermediates as thioesters) has been proposed as a mode of action of ACVS (17), we have been especially interested in conserved cysteine residues within the homologous regions or adjacent stretches. Unex- pectedly, the four conserved cysteine thiol groups found in the two bacterial peptide synthetase multienzymes, tyrocidine synthetase 1 and gramicidin synthetase 1 (31), are missing in the ACVS sequence. However, sequences bearing similarity to an active site peptide involved in valine-binding in grami- cidin synthetase 2 (LGGHXRAM, Ref. 36) have been located in each of the domains. The mode of attachment of valine to this peptide remains unknown. Interestingly, seryl residues are found within these motifs in ACVS (Fig. 5C). Some

A. nidulans ACV Synthetase 12653

L A C T A T E D E H Y D R O G E N A S E I140 koa) A L D O L A S F 1160 XD.1

10

w F E R R I T I N ( 4 4 0 kDs1

A C V S &%,

1.8 2.0 2.2 2 4 2.6 2.8 3 0 Log molecular welghl

FIG. 6. Molecular mass determination of ACVS by FPLC.

1 2 3 4 5 6

FIG. 7. Molecular mass determination by native polyacryl- amide gel electrophoresis. Lane 1, bovine serum albumin (66,132, 198, 264, and 330 kDa), lane 2, phosphorylase B (375 kDa), lane 3, catalase (240 kDa), lane 4, ferritin (220 and 440 kDa), lane 5, thyro- globulin (335 and 670 kDa), lane 6, ACVS. The size of ACVS has been estimated from a linear plot of relative mobilities (m,) of peak positions against log molecular mass.

I 2 koa

ACVS- -205

-116

FIG. 8. Visualization of ACVS as a glycoprotein by affinity detection with ConA. ACVS was subjected to SDS-PAGE and transferred to a nitrocellulose sheet, which was incubated first with ConA and then with peroxidase (see "Experimental Procedures"). Lane I , 150 ng of ACVS, incubated 15 min a t room temperature in the absence of a-glucosidase. Lane 2,150 ng of ACVS, incubated with cy-glucosidase for 15 min a t room temperature. Molecular mass mark- ers used were myosin (205 kDa) and 8-galactosidase (116 kDa).

similarity to 4'-phosphopantetheine attachment sites de- scribed for polyketide synthases (i.e., DSL) can be noted and one could speculate on possible pantetheine-bound acyl inter- mediates; this may reflect, for example, the attachment of multiple cofactors to ACVS, resulting in a modified mecha- nism for the thiotemplate pathway to polypeptides. As yet, we have been unable to establlsh the precise pantetheme content of the A. nidulans enzyme. The presence of this cofactor, nevertheless, has been shown for the ACVS of Ceph- alosporium acremonium:' by microbiological assay, for which

:I U. Fink and H. von Dohren, unpublished results.

RAT THIOESTERASE 87 LPIIQDKAFAFFGHSFGSYIALITALLLKEK 117 DUCK THIOESTERASE 76 *KDL*E*P**L******'FVSYAL*VH**** 106 grsT ACVS 3611 IQP..'GPYH*I*W***GT"MEISRQ"VGL 3639

81 IQPLINIP"**L*H*M*AL*SFEL*RTIRQ* 111

FIG. 9. Alignment of related sequences between rat and duck thioesterases, gramicidin synthetase T, and the COOH- terminal region of ACVS.

data indicate a single moiety per ACVS molecule. It is note- worthy that the sequence implicated in pantetheine attach- ment is also present in gramicidin synthetase 1 and tyrocidine synthetase 1, although in both of these multienzymes no pantetheine has yet been found.

Between the first and second ACVS domains, an additional potential cofactor attachment site has been detected that bears some similarity to sequences found in gramicidin syn- thetase 2 and tyrocidine synthetase 2 (30) (Fig. 50) . If mul- tiple cofactors are present in ACVS, this additional site may serve to transport the completed ACV tripeptide to a thioes- terase-releasing site. A sequence recently identified in the gramicidin S gene cluster (gr sT) that shares homologies with avian medium chain thioesterases (31) and two genes of the Streptomyces bialaphos biosynthetic cluster (38) has been aligned with the ACVS sequence, resulting in the identifica- tion of a carboxyl-terminal region that also shows similarities to the thioesterase active site region, GXSXG (Fig. 9). Thus, the required thioesterase function may be an integral part of the multienzyme as indicated by the activity of the purified protein with regard to ACV synthesis (17). However, the present state of characterization cannot exclude the function of minor proteins that may have escaped electrophoretic detection and could significantly increase the rate of peptide formation. In this regard, evidence for a 30-kDa protein associated with ACV synthetase from Streptomyces clavuli- gerus has been obtained recently (39).

ACVS has also been identified as a glycoprotein by means of affinity blotting with Con A. This finding is in accord with the reported localization of P. chrysogenum ACVS activity in 200-nm vesicles (Golgi-derived) (37) and suggests therefore that the A. nidulans enzyme is also compartmentalized rather than free in the cytoplasm.

Analysis of proteolytic digestion products of ACVS confirm the reading frame deduced from the acuA DNA sequence and help to confirm 1) that the ACVS protein has not been subject to excessive degradation during preparation as evidenced by the distribution of subtilisin-derived polypeptides in relation to the DNA derived protein sequence and 2) that it appears unlikely that the domains are separated by introns, as one of the polypeptides isolated corresponds to an interdomain re- gion. With regard to the latter, the revised size of ACVS of 422 kDa is now in accord with that predicted from translation of the gene sequence.

That such a large protein is required for the production of the ACV tripeptide may be due to the number of catalytic steps that are carried out, such as the recognition of the three amino acids, their subsequent adenylation, peptidation, epi- merization, and release of product.

The sequence of ACVS should permit molecular dissection of the functions of this multifunctional peptide-synthesizing protein and facilitate analyses of the evolutionary relation- ships between such proteins.

Nimmo, and G. Walker (University of Glasgow) for help with protein A C ~ ~ I W W ~ ~ & I I C C I C ~ ~ W c wish LU Lllulrh PluL J. Cuggilm, Dl. 11.

purification, fractionation, and sizing; Prof. J. Fothergill and B. Dunbar (University of Aberdeen) for help with amino acid sequenc- ing; and Prof. G. Turner (University of Sheffield) for making available A. nidulans cosmid clone CX35. We also thank Dr. R. Weckermann (Technische Universitat Berlin) for pointing out possible pantetheine

12654 A. nidulans ACV Synthetase

attachment sites in related peptide synthetases and for help in se- 17. van Liempt, H., von Dohren, H., and Kleinkauf, H. (1989) J. quence alignments. Finally, H. D. acknowledges Drs. L. van der Voort Biol. Chem. 264,3680-3684 and A. Veenstra, Gist-brocades NV (Netherlands), for their work in 18. Kleinkauf, H., and von Dohren, H. (1987) Annu. Reu. Microbiol. the amino-terminal sequencing of subtilisin-derived fragments. 41,259-289

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