biosynthesis of ansatrienin (mycotrienin) and naphthomycin : identification and analysis of two...

Eur. J. Biochem. 261, 98±107 (1999) q FEBS 1999

Biosynthesis of ansatrienin (mycotrienin) and naphthomycinIdentification and analysis of two separate biosynthetic gene clusters in

Streptomyces collinus TuÈ 1892

Shuo Chen1, Daniel von Bamberg2, Valerie Hale1, Michael Breuer2, Birgit Hardt2, Rolf MuÈ ller2, Heinz G. Floss3,

Kevin A. Reynolds1 and Eckhard Leistner2

1Institute for Structural Biology and Drug Discovery & Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, USA;2Institut fuÈr Pharmazeutische Biologie, Rheinische Wilhelms-UniversitaÈt, Bonn, Germany; 3Department of Chemistry, University of Washington,

Seattle, WA USA

The polyketide chains of the two ansamycin antibiotics, ansatrienin (mycotrienin) and naphthomycin produced by

Streptomyces collinus are assembled using 3-amino-5-hydroxybenzoic acid (AHBA) as a starter unit. The gene

encoding AHBA synthase, an enzyme which catalyzes the final step of AHBA biosynthesis in the recently

discovered aminoshikimate pathway, has been used to identify two separate antibiotic biosynthetic gene clusters in

S. collinus. In one of these clusters, analysis of approximately 20 kb of contiguous sequence has revealed both a

cluster of six genes presumed to play a role in the AHBA pathway and the beginning of a polyketide synthase (PKS)

gene containing an acyl ACP ligase domain. This domain is likely responsible for loading AHBA onto the PKS. This

gene cluster also contains chcA, encoding the enzyme 1-cyclohexenylcarbonyl CoA reductase, which is essential for

the biosynthesis of the cyclohexanecarboxylic acid moiety of ansatrienin from shikimic acid, and a peptide

synthetase. This gene cluster thus seems to control the biosynthesis of ansatrienin, which contains a side chain

of N-cyclohexanecarbonyl-d-alanine esterified to the macrocyclic lactam backbone. In the putative naphthomycin

biosynthetic gene cluster approximately 13 kb of contiguous sequence has revealed a second set of the genes

required for AHBA biosynthesis. In addition the end of a polyketide synthase and a gene putatively involved in

termination of the chain extension process, formation of an intramolecular amide bond between the AHBA nitrogen

and the carboxyl group of the fully extended polyketide chain, have been identified. Thus, despite commonality in

biosynthesis, the ansatrienin and naphthomycin biosynthetic gene clusters show clear organizational differences and

carry separate sets of genes for AHBA biosynthesis.

Keywords: ansatrienin, naphthomycin, aminohydroxybenzoic acid, cyclohexanecarboxylic acid, Streptomyces

collinus, polyketide biosynthesis.

Ansamycin antibiotics including rifamycin, naphthomycin,ansatrienin (mycotrienin) are structurally related, containing amacrocycle of polyketide origin with an amide linkage to eithera naphthalenic or benzenic aromatic moiety (Fig. 1) [1]. Feedingexperiments with rifamycin (produced by Amycolatopsis medi-terranei), ansatrienin [2] and naphthomycin [3] (produced byStreptomyces collinus) have demonstrated that the polyketidechain of these antibiotics is assembled from acetate andpropionate units in a manner typical of that catalyzed by amultifunctional type I polyketide synthase [4]. The starter unit

for these antibiotics is 3-amino-5-hydroxybenzoic acid (AHBA)derived by the newly discovered aminoshikimate pathway, aprocess which parallels the initial steps of the shikimate pathway(Fig. 2) [1]. 5-deoxy-5-amino-3-dehydroshikimic acid (amino-DHS) formed in this pathway is converted to AHBA by theaction of AHBA synthase [5].

There is considerable precedent that the genes involved inassembly of the antibiotics from simple carbon precursors areclustered with genes providing self-resistance and the regulatorygenes that coordinate expression of the biosynthetic genes[6±8]. Genes involved in pathways that convert primarymetabolites to precursors used for secondary metabolism oftenalso appear to be located within these gene clusters [8±11]. Inparticular, it has been shown recently that the genes involved inAHBA biosynthesis in A. mediterranei are clustered with theother rifamycin biosynthetic genes [12]. Other ansamycinbiosynthetic gene clusters might therefore be expected to includeAHBA biosynthetic genes.

S. collinus provides an unusual system in which to test thishypothesis as it produces two ansamycin antibiotics, ansatrieninA (mycotrienin) and naphthomycin C [13,14]. Thus, thisorganism may contain either one set of AHBA biosyntheticgenes, which provides AHBA for both processes, or individualsets of AHBA biosynthetic genes within each ansamycin gene

Correspondence to K. A. Reynolds, 800 E. Leigh St., Suite 212B,

Richmond, VA 23219, USA. Fax: + 1804 8273664,

E-mail: [email protected]

Abbreviations: AHBA, 3-amino-5-hydroxybenzoic acid; aminoDHS,

5-deoxy-5-amino-3-dehydroshikimic acid;PKS, polyketide synthase; ACP,

acyl-carrier protein; CHC, cyclohexanecarboxylic acid; CHC-CoA, CoA

thioester of cyclohexanecarboxylic acid; DAHPs, deoxy-d-arabino-hept-2-

ulosonate 7-phosphate synthase; EPSPs, 3-enolpyruvylshikimate

5-phosphate synthase.

Notes: S.C. and D.B. contributed equally to this project. This paper is

dedicated to the memory of Professor Claire Berg

(Received 14 September 1998, revised 15 December 1998, accepted

12 January 1999)

q FEBS 1999 AHBA Biosynthetic gene clusters in S. collinus (Eur. J. Biochem. 261) 99

cluster. Ansatrienin A also contains an unusual cyclohexane-carboxylic acid (CHC) moiety attached via a d-alanine residueto the polyketide chain [15,16]. This CHC is derived fromshikimic acid by a pathway that involves a series of reductionand dehydration steps, with some or all of the intermediatesactivated as coenzyme A thioesters (Fig. 3) [17,18]. The CHCbiosynthetic genes might be predicted to be clustered with theansatrienin biosynthetic genes. As inactivation of chcA, encod-ing a reductase involved in CHC biosynthesis, results in amutant unable to produce both CHC and ansatrienin A and B,the association of this gene with AHBA synthesis and polyketide

synthase (PKS) genes would provide strong evidence for theassignment of this cluster as the ansatrienin biosynthetic genecluster [19]. A clear affiliation between CHC biosynthesis andansatrienin biosynthesis, however, has not previously beendemonstrated. For instance, ansatrienin analogs have beenisolated in which the CHC moiety is replaced with isobutyricand methylbutyric acid moieties [20], compounds which arecommonly used to initiate fatty acid biosynthesis in strepto-mycetes [21]. Conversely, CHC has been observed to be used asstarter unit in fatty acid biosynthesis in micro-organisms,including S. collinus [19].

In this work rifK [12], the gene encoding AHBA synthase inA. mediterranei, and chcA, a gene encoding a reductive enzymein the CHC pathway, were used as probes to detect theS. collinus ansatrienin and naphthomycin biosynthetic geneclusters. One gene cluster was shown to contain genes encodinga type I modular polyketide synthase, the genes required forAHBA biosynthesis, and chcA as well as other genes potentiallyinvolved in CHC biosynthesis. This cluster therefore probablyencodes ansatrienin biosynthesis. A second gene cluster, likelyinvolved in naphthomycin C biosynthesis, has been identifiedand shown to contain genes encoding another type I modularpolyketide synthase and a second set of the AHBA biosyntheticgenes. The organization of the individual AHBA genes in eachcluster differs, as does the relative positioning of these geneswith respect to the polyketide synthase genes.

MATERIALS AND METHODS

Bacterial strains, plasmids, and cosmids

S. collinus TuÈ 1892 was a gift from Professor Axel Zeeck(UniversitaÈt GoÈttingen, Germany) and A. mediterranei S699from Professor Giancarlo Lancini (Lepetit Research Laboratory,Gerenzano, Italy). Escherichia coli AB2834/pIA321, a geneti-cally engineered overproducer of shikimate dehydrogenase, wassupplied by Professor John R. Coggins (University of Glasgow,UK). Dr B. Schoner (Eli Lilly and Co.) provided the cosmidvector pOJ446 for cosmid library construction and Dr LeonardKatz (Abbott Laboratories) provided the eryA ketosynthaseprobe.

A second cosmid library of S. collinus in pDUAL3 wasprovided by Dr Claire Berg (University of Connecticut, USA).Subcloning steps were performed using the plasmid pBluescriptII KS(-) and E. coli strain XL-1 Blue MRF 0 (Stratagene).Protein expression studies were carried out using vector pRSETB (Invitrogen) and E. coli BL21(DE3)pLysS (Stratagene).

Growth of organisms

E. coli strains carrying plasmids or cosmids were grown inliquid Luria-Bertani (LB) medium or on solid LB medium(1.5% agar) at 37 8C and were selected with ampicillin(100 mg´mL±1) or apramycin sulfate (100 mg´mL±1) [22].S. collinus and A. mediterranei were grown in yeast extract-malt extract medium (YEME) at 28 8C [23].

Isolation and DNA manipulation

General methods for DNA manipulation and isolation wereperformed as previously described for Streptomycetes [23] andfor E. coli [22]. Plasmid DNA isolation was carried out usingQiagen ion exchange columns (Qiagen). Restriction digests withendonucleases were carried out following the manufacturer'sprotocols. DNA fragments were isolated from agarose gels withthe QIAquick gel extraction kit (Qiagen).

Fig. 1. Structures of the ansamycin antibiotics ansatrienin A and

naphthomycin C, produced by Streptomyces collinus TuÈ 1892 and

rifamycin B produced by Amycolatopsis mediterranei S699.

100 S. Chen et al. (Eur. J. Biochem. 261) q FEBS 1999

Gene library construction and probing

S. collinus chromosomal DNA was partially digested withSau3AI, dephosphorylated and ligated into cosmid vectorpOJ446, previously digested with HpaI, dephosphorylated andrestricted with BamHI. The ligation products were packagedwith Gigapack III Gold (Stratagene) and transduced into E. coliXl-1 Blue MRF 0. The resulting cosmid library and a pDUAL 3library of S. collinus DNA were then analyzed by Southernhybridization for both AHBA synthase genes and for chcA. The0.9-kb AHBA synthase probe was obtained by amplifyingA. mediterranei genomic DNA using the following primers 8(5 0-CCTGCGTGGCCGCAGTACGAC-3 0) and 10 (5 0-GATGCGGAACATGGCCATGTA-3 0). The chcA probe was obtained by aBamHI, NdeI double digest of pPW4 [19]. Cosmid clonesidentified in this manner were then BamHI restricted anddivided into two groups, those containing a 1.7-kb and those

with a 3.4-kb BamHI fragment which hybridized to the AHBAsynthase gene probe.

DNA sequencing and computer assisted sequence analysis

Cosmid clones identified by probing of the cosmid library weredigested and subcloned as smaller fragments. These were thensequenced using the Amplitaq dye-terminator sequencingsystem (Perkin Elmer) and run on an automated DNA sequencer(Applied Biosystems, model 377). DNA and deduced proteinsequence analyses and alignments were carried out usingMacVector and DNASTAR seqman/megalign programs. Incases of related proteins of different lengths the amino acidsequence identities were calculated based only on the region ofoverlap with the shorter protein. Deduced amino acid sequencedata were compared with the National Center for Biotechnology

Fig. 3. Formation of CHC-CoA (the coenzyme

A thioester of cyclohexanecarboxylic acid) in

S. collinus. Proposed (#) and determined (*) roles

of gene products from the putative ansatrienin

biosynthetic gene cluster are indicated.

Fig. 2. Formation of AHBA (3-amino-5-hydroxybenzoic acid) by the aminoshikimate pathway (A), compared to the shikimic acid biosynthetic pathway

(B). Proposed (#) and determined (*) roles of gene products from the rifamycin (A. mediterranei), ansatrienin and putative naphthomycin (S. collinus) gene

clusters are indicated. The so-called phosphatase (napH, ansH, rifM), kinase (ansB, napI, rifN ) and oxidoreductase (ansG, napG, and rifL) genes located in the

AHBA biosynthetic gene clusters also play a role in this process.


Information (NCBI) database using the BLAST search at theweb site: HTTP://www.ncbi.nlm.nih.gov/BLAST.

Expression of the AHBA synthase in E. coli

The putative AHBA synthases identified in S. collinus wereexpressed in E. coli as His6-tagged proteins. Using cosmidDNA as template the entire genes ansF and napF were amplifiedby PCR using Pfu DNA polymerase (Stratagene). The forwardprimers were designed to introduce both a BglII restriction site(underlined) upstream of the start codon and a NdeI site(CATATG) overlapping the start codon (boldface). Theseprimers for ansF and napF were 5 0-GAGATCTGCATATGAG-CAGTGGTGTGCAACTGGGC-3 0 and 5 0-CAGATCTGCA-TATGAACGCGCGACCGGCACCGGA-3 0, respectively. Thereverse primers designed to introduce a KpnI site (underlined)downstream of the stop codon were 5 0-CGGTACCGCCGCCGTCGATGTCGGCCAC-3 0 (ansF) and 5 0±CGGTACCAGCCCCACCACGGCGGCCCGTAC-3 0 (napF). The PCR products,1200-bp ansF and 1204-bp napF, were cloned into pCR-Script(Stratagene) following the manufacturer's protocol, digestedwith BglII and KpnI, and subsequently cloned into the BamHIand KpnI sites of pRSET B (a T7 expression vector). Theresulting plasmid was transformed into E. coli BL21(DE3)-pLysS. The resulting E. coli BL21(DE3)pLysS coloniesharboring the pRSET B/AHBA constructs were used toinoculate 10 mL of LB medium containing ampicillin(100 mg´mL±1) and chloramphenicol (34 mg´mL±1) and grownovernight at 37 8C. One milliliter of these overnight cultures wasused to inoculate 100 mL LB medium containing the sameantibiotics in identical concentrations and additionally 1 msorbitol and 2.5 mm betaine. The cultures were grown at 28 8Cwith shaking (180 r.p.m.) and after 8 h induced by addition ofisopropyl thio-b-d-galactoside to a final concentration of0.1 mm. The cells were incubated for a further 24 h andharvested. Cell-free extracts were prepared and analyzed forAHBA synthase activity by an inverse isotope dilution assay aspreviously described but using 40 mg of [7-13C] AHBA [1]

Purification of the recombinant AHBA synthases

The recombinant N-terminal His6-tagged AnsF and NapF werepurified using nickel-nitrilotriacetic acid affinity chromatogra-phy following the manufacturer's protocol for column purifica-tion under native conditions (Qiagen).

Nucleotide sequence accession number

The nucleotide sequence of the biosynthetic genes has beensubmitted to GenBank under accession numbers AF131877,AF131878 and AF131879.

RESULTS

Nucleotide sequence of the two separate clusters of AHBAbiosynthetic genes

A 900-bp PCR product derived from the AHBA synthase gene(rifK) of A. mediterranei was used in heterologous hybridizationexperiments with two cosmid libraries of S. collinus DNA. Thisapproach identified overlapping cosmid clones for two separategene clusters containing genes with sequence homology to rifK(Fig. 4 and Table 1) [5]. As shown in Table 2 the deducedamino acid sequence of one of the AHBA synthases in theputative naphthomycin biosynthetic gene cluster (NapF) exhibited

higher identity (82%) to the rifK gene product than did thededuced amino acid sequence of the corresponding gene (ansF)in the ansatrienin biosynthetic gene cluster (69%). The basis forassignment of the two gene clusters is described below. Bothgene products contain the conserved active site lysine residueassociated with the A. mediterranei AHBA synthase and otherpyridoxal phosphate-dependent enzymes [5].

The well-established precedent for clustering of relatedbiosynthetic genes in actinomycetes [6±8] suggested that othergenes involved in AHBA biosynthesis in S. collinus may beclustered with ansF and napF. The first three steps of the AHBAbiosynthetic pathway ± preceding the conversion of aminoDHSto AHBA ± are very similar to the first three steps of theshikimate pathway. Thus the DNA surrounding ansF and napFwas sequenced and analyzed for genes which may encodeproteins with similarity to enzymes involved in the shikimatepathway.

In this manner two genes, ansI and napD, encoding proteinswith sequence homology to higher plant type 3-deoxy-d-arabino-hept-2-ulosonate 7-phosphate synthases (DAHPs) were identi-fied. Approximately 42% amino acid sequence identity wasobserved for both AnsI and NapD with the AroG ofLycopersicon esculentum and Arabidopsis thaliana [24,25].Identities of 57% (AnsI) and 35.4% (NapD) were observedwith the DAHPs involved in phenazine biosynthesis inPseudomonas fluorescens [26]. Amino acid sequence similarityhas recently been reported between various plant type DAHPsand the rifH gene product of A. mediterranei, leading to thesuggestion that RifH is involved in catalyzing the first step ofthe AHBA biosynthetic process [12]. This step involvesconversion of phosphoenolpyruvate and erythrose 4-phosphateto aminoDAHP (Fig. 2), although the source of the nitrogen inthis step and the manner in which it is incorporated into theproduct are as yet undetermined. Similar roles can be envisionedfor AnsI and NapD.

Two genes (ansA and napC) which encode proteins withsequence similarity to known dehydroquinate synthases (39%and 43% identity to AroB of E. coli, respectively) were alsoidentified in close proximity to ansF and napF (Fig. 4, Tables 1and 2) [27]. The second step in the AHBA biosynthetic pathway,conversion of aminoDAHP to aminodehydroquinate (Fig. 2) isanalogous to the reaction carried out by dehydroquinatesynthase in the shikimate pathway [1]. It is proposed thatthese two gene products are involved in formation ofaminodehydroquinate. In A. mediterranei this reaction isthought to be catalyzed by RifG which has strong sequencesimilarity with both AnsA and NapC and dehydroquinatesynthases [12].

Two additional genes which encode proteins with homologyto enzymes of the shikimate pathway were also observed. TheansE gene product exhibits sequence similarity to type IIdehydroquinate dehydratases, e.g. from Streptomyces hygrosco-picus var ascomyceticus (27% identity) [28]. The third step inthe AHBA biosynthetic pathway is analogous to the reactioncatalyzed by a dehydroquinate dehydratase, involving adehydration of aminodehydroquinate to aminodehydroshiki-mate, and is likely catalyzed by AnsE (Fig. 2). No obviousdehydroquinate dehydratase gene was present in the sequencedregion of the putative naphthomycin gene cluster. It is reason-able to propose that this gene lies outside of the region directlysurrounding napF, because in the rifamycin biosynthetic genecluster (which exhibits a similar organization of the AHBAbiosynthetic genes) rifJ, which encodes a protein with sequencesimilarity to both the ansE gene product and type IIdehydroquinate dehydratases, is located 30 kb downstream


from the other AHBA synthesis genes [12]. Insertionalinactivation experiments have demonstrated that rifJ is involvedin AHBA formation. The napE gene product exhibits similarityto shikimate dehydrogenases (31% identity to the aroE geneproduct of Pseudomonas aeruginosa). No homolog of the genewas identified in the sequenced region of the ansatrieninbiosynthetic gene cluster. The AHBA biosynthetic pathway doesnot contain an analogous step to that catalyzed by shikimatedehydrogenase in the shikimate pathway and the role of NapE inAHBA biosynthesis is unclear. Interestingly, rifI in the rifamycin

biosynthetic gene cluster encodes a similar protein withhomology to shikimate dehydrogenases; the protein expressedfrom it catalyzes the 3-dehydrogenation of shikimate, amino-shikimate and aminoquinate, but not quinate (R. MuÈller & H. G.Floss, unpublished results).

Located downstream from napF are three genes, napG, napHand napI that show sequence homology to rifL, rifM and rifN ofthe rifamycin gene cluster, respectively (Fig. 4, Table 2).Homologues of the first two of these genes, ansG and ansHare also located directly downstream of ansF. A homologue of

Fig. 4. Organization of the AHBA biosynthetic

region of the ansatrienin (A) and putative

naphthomycin (B) gene clusters. The AHBA

biosynthetic region of the rifamycin gene cluster is

also shown (C). Each arrow represents an open

reading frame (ORF) except napA2. Patterned

arrows indicate genes involved in AHBA bio-

synthesis. The same pattern is used for homo-

logous genes in each of the three clusters. The

direction of transcription and the relative size of

the ORFs deduced from the nucleotide sequence

are shown. The proposed functions of each ORF

are given in Table 1.

Table 1. Proposed functions of ORFs in the proposed ansatrienin and naphthomycin biosynthetic gene cluster. The function of chcA in

cyclohexanecarboxylic acid biosynthesis has previously been established [19]. The demonstration in the current study that the recombinant AnsF/NapF

catalyzes the AHBA synthase reaction in vitro supports the proposed role for the corresponding gene.

Gene Sequence similarity Proposed function

ansA Dehydroquinate synthase AHBA Biosynthesis

ansB Kinase AHBA Biosynthesis

ansC Peptide synthetase CHC-alanine ligation

ansD Unknown

ansE Dehydroquinate dehydratase AHBA Biosynthesis

ansF AHBA synthase AHBA Biosynthesis

ansG Oxidoreductase AHBA Biosynthesis

ansH Phosphatase AHBA Biosynthesis

ansI DAHP synthase AHBA Biosynthesis

ansJ EPSP synthase CHC Biosynthesis

ansK Coumarate CoA ligase CHC Biosynthesis

ansL Acyl CoA dehydrogenase CHC Biosynthesis

chcA 1-Cyclohexenylcarbonyl CoA reductase CHC Biosysthesis

ansM NADH-dependent flavin oxidoreductase CHC Biosynthesis

napA1 Arylamine acyltransferase Naphthomycin ring closure

napB Non-heme oxygenase/halogenase Naphthomycin modification

napC Dehydroquinate synthase AHBA Biosynthesis

napD DAHP synthase AHBA Biosynthesis

napE Shikimate/quinate dehydrogenase AHBA Biosynthesis

napF AHBA synthase AHBA Biosynthesis

napG Oxidoreductase AHBA Biosynthesis

napH Phosphatase AHBA Biosynthesis

napI Kinase AHBA Biosynthesis


napI/rifN, ansB, was found in a different location within theansatrienin biosynthetic gene cluster (Fig. 4). The rifL, napG,and ansG gene products all show homology to glucose-fructoseoxidoreductases, e.g. of Zymomonas mobilis [30], whereas thenapH, ansH and rifM gene products show similarity tophosphatases involved glycolate oxidation [31]. The napI,ansB and rifN gene products all show homology to a glucosekinase involved in glucose catabolite repression in Streptomy-ces coelicolor [32]. Gene inactivation experiments in A. medi-terranei have established that the rifL, rifM and rifN genes areall involved in AHBA formation, but their specific functions areat present unknown. (R. MuÈller, H. G. Floss, unpublishedresults.)

The organization of the AHBA biosynthetic genes in theputative naphthomycin polyketide synthase gene cluster isvirtually indistinguishable from that in the rifamycin cluster(Fig. 4). In comparison the AHBA synthase genes in theansatrienin biosynthetic gene cluster have a quite differentorganization, the only similarity being the location of the so-called oxidoreductase (ansG) and phosphatase (ansH) down-stream of the AHBA synthase gene (ansF). In addition, thegenes involved in AHBA biosynthesis from the putativenaphthomycin biosynthetic gene cluster exhibit significantlyhigher sequence similarity with the corresponding genes of therifamycin cluster than they do with those in the ansatrienincluster (Table 2).

AHBA synthase expression, purification and characterization

Verification of the role of the putative AHBA synthase genesansF and napF in AHBA biosynthesis was obtained bydemonstrating that the recombinant proteins expressed fromthese genes exhibited the appropriate catalytic activity. A PCRstrategy was used to introduce the ansF and napF genes into thevector pRSET B for expression as His6-tagged fusion proteinsunder the control of the T7 promoter. E. coli BL21(DE3)pLysSharboring either of the two expression constructs was thengrown under conditions optimized for obtaining soluble protein.Expression of the recombinant AHBA synthases was subse-quently analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS/PAGE). Crude cell-free extracts obtainedfrom the ansF and napF expression clones exhibited strongprotein bands at approximately 45 kDa and 43 kDa, respectively(Fig. 5). These bands were not seen in SDS/PAGE analysis ofextracts from control cultures of E. coli BL21(DE3)pLysScarrying pRSET B without insert DNA. SDS/PAGE analysisof the proteins purified by affinity chromatography on a nickelcolumn revealed single major bands corresponding to molecularmasses of 45 kDa and 43 kDa for AnsF + His6 and NapF +His6, respectively (Fig. 5). These molecular masses are slightlylower than the predicted values (46.5 kDa for AnsF + His6 and

45.8 kDa for NapF + His6). A similar observation has beenmade with the recombinant His6-tagged AHBA synthase fromA. mediterranei.

AnsF and NapF were shown to be catalytically competentAHBA synthases using a GC-MS-based inverse isotope dilutionassay. Crude cell-free extracts of E. coli expressing ansF andnapF both produced 27 mg and 26 mg of unlabeled AHBA fromaminoDHS, respectively. A parallel incubations with an extractfrom E. coli BL21(DE3)pLysS/pRSET B showed no detectableconversion of aminoDHS into AHBA.

Nucleotide sequence of a set of genes putatively involved inCHC biosynthesis

The previously identified chcA gene [19] was located within aset of five open reading frames that lie downstream of ansI(Fig. 4, Table 1). The chcA gene product encodes ChcA whichhas been shown to catalyze in vitro three of the a,b-enoyl CoAreduction steps that occur in the biosynthesis of CHC (Fig. 3).Insertional inactivation of chcA has previously been shown toproduce a S. collinus mutant (CD1075) incapable of synthesiz-ing either CHC or ansatrienin A [19]. The clustering of the

Table 2. Comparisons of predicted amino acid sequence identity between the AHBA biosynthetic genes of the rifamycin gene cluster and the ansatrienin

and naphthomycin gene clusters.

Proposed enzymes ANS/NAP (%) ANS/RIF (%) NAP/RIF (%)

AHBA synthase 73 69 82

Phoaphatase 72 75 82

(amino)dehydroquinate synthase 75 74 76

(amino)DAHP synthase 33 38 71

Oxidoreductase 55 42 70

Kinase 46 53 63

(amino)quinic/shikimic acid dehydrogenase N/A N/A 72

(amino)dehydroquinate dehydratase N/A 51 N/A

Arylamine acyltransferase N/A N/A 58

Fig. 5. SDS/PAGE analysis of the recombinant AHBA synthases. Lanes:

(a), crude cell-free extract of E. coli expressing napF; (b), crude cell-free

extract of E. coli expressing ansF; (c), NapF sample purified via His6-

binding resin; (d), AnsF sample purified via His6-binding resin; (e), RifK

(AHBA synthase from A. mediterranei, loaded for size comparison) sample

purified with His6-binding resin; (f), molecular weight markers (phosphory-

lase B, 97 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa;

carbonic anhydrase, 31 kDa).


AHBA biosynthetic genes suggested that the genes involved inCHC biosynthesis may be similarly clustered. Thus the ORFssurrounding chcA were analyzed to determine if their deducedamino acid sequence provides an indication as to a potential rolein CHC biosynthesis.

AnsJ was found to have amino acid sequence similarity to 3-enolpyruvylshikimate 5-phosphate synthase (EPSPs), an enzymeinvolved in the conversion of shikimic acid to chorismic acid.Chorismic acid is not a precursor in CHC biosynthesis, and thesequence similarity to known EPSPs enzymes is relatively weak(less than 25% similarity) [33]. AnsJ may not be a true EPSPs,but rather, it may catalyze the first committed step of the CHCpathway, conversion of shikimate or its 3-phosphate to (3R, 4R)-3,4-dihydroxycyclohexa-1,5-dienecarboxylic acid (Fig. 4). Thesubstrate for both of these reactions may well be the same as ithas yet to be established if shikimic acid or shikimate-3-phosphate is the substrate for the first step in CHC biosynthesis.AnsK has strong amino acid sequence identity to a number of 4-coumarate CoA ligases (29% identity with the Mycobacter-ium leprae enzyme) and 2,3-dihydroxybenzoyl: AMP ligases(44% identity to the enzyme of E. coli) [34]. AnsK is proposedto catalyze the conversion of 4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid to the corresponding CoA thioester(Fig. 3). There are clear structural similarities between thesubstrate for this reaction and the substrates of the coumarate:CoA ligase and dihydroxybenzoyl: AMP ligases. AnsK containsregions of homology corresponding to the three putativeadenylate binding motifs of the acyl-adenylate/thioester enzymesuperfamily [35].

AnsL has approximately 30% amino acid sequence similarityto acyl CoA dehydrogenases involved in fatty acid degradation.AnsM (downstream of chcA) has some amino acid sequencesimilarity to NADPH-dependent oxidoreductases (41% to theyeast NADPH-dependent flavin oxidoreductase). The physiolo-gical role of these NADPH-dependent oxidoreductases iselusive, although recent studies suggest they may be involvedin a novel dismutation reaction and that NADPH is itsphysiological reductant (the in vivo electron acceptor isunknown) [36]. An NADPH-FAD oxidoreductase gene, vlmR,has recently been found clustered with the valinomycinbiosynthetic genes in Streptomyces viridifaciens [37]. Theassignment of putative roles for AnsL and AnsM in CHCbiosynthesis based on these sequence similarities is less obviousthan those of AnsJ and AnsK, but does not preclude them fromplaying a role.

Partial nucleotide sequences of the presumed ansatrienin andnaphthomycin polyketide synthase and polyketide modifyinggenes

Southern hybridization experiments with an eryA ketosynthaseprobe indicated that a polyketide synthase gene was locatedupstream of ansG. This preliminary observation was supportedby sequence analysis which revealed the 5 0 end of a loadingdomain for a polyketide synthase which had 64% and 54%identity to the loading domains of the rifamycin and rapamycinpolyketide synthases, respectively [12].

In contrast, the 3 0 end of a type I polyketide synthase genewas observed upstream of napG (Fig. 4). Slightly more than onemodule of this polyketide synthase was sequenced. The presenceof a dehydratase, a ketoreductase and an acyl transferase domainwithin this module are consistent with a role in catalyzing thelast chain extension step in the biosynthesis of naphthomycin C[14]. However, it is also possible that this module is involvedin a different step of naphthomycin biosynthesis. Located

downstream of this PKS gene is napA1, which encodes a proteinwith a high degree of sequence similarity to the gene product ofrifF from the rifamycin biosynthetic gene cluster (Fig. 4) and toarylamine : acetyl-CoA acetyltransferases, detoxifying enzymeswhich acetylate the amino group of aromatic amines [38]. Theproposed mechanism for arylamine acetyl transferases involvestransfer of the acyl group of acetyl CoA to a conserved cysteineresidue on the protein prior to formation of the amide bond. Thededuced amino acid sequences of NapA1 and RifF also containthis cysteine residue within a conserved peptide region. It isproposed that the carboxy terminus of the fully extendedpolyketide chain may be transferred from the polyketidesynthase to the conserved cysteine residue and then an amidebond is formed with the amino group of the aromatic moiety. Asimilar role has been proposed for RifF in rifamycin biosynthe-sis and has recently been substantiated by gene inactivationexperiments (T. W. Yu, Y. Shen, B. S. Moore, C. R. Hutchinson& H. G. Floss, unpublished results).

Located between the PKS and napA1 there is a 600-bp regionof DNA. The first 280 bp exhibit the typical codon usagepreference observed for Streptomyces ORFs and would encode apeptide with 54% identity to the gene product of napA1. There isno identifiable start or stop codon for this region and it is thusunlikely that there is a napA2 gene product. The second half ofthe 600-bp region contains a 300-bp ORF that encodes an ACP(ACP2). This ACP has a clear start and stop codon and aputative ribosomal binding site upstream of the start codon, andshows clear end to end sequence homology with various ACPdomains from type I polyketide synthases (data not shown). Thesequence identity between ACP2 and ACP1 is 55%. Nosequence similarity between this ACP and type II PKS ACPswas observed except a few amino acids around the presumedphosphopantethine attachment site. PCR experiments confirmedthat the arrangement of the napA2 and ACP2 sequences betweenthe PKS and napA1 observed on the cosmid clone was alsopresent in the chromosomal DNA. For instance, a PCR productof the predicted size (4.3 kb) was obtained using the cosmidDNA and PCR primers based on a region of the acyl transferasedomain of the PKS and a region of DNA between napA1 andACP2. A product of the same size was obtained usingchromosomal DNA as a template (data not shown). The role,if any, of the region of DNA containing napA2 and ACP2, in thenaphthomycin biosynthetic process is unclear.

The predicted amino acid sequence of AnsC shows significantsimilarity with a wide variety of peptide synthetases (38% and25% amino acid identity to an actinomycin peptide synthetaseand a peptide synthetase of Amycolatopsis mediterranei,respectively) and with the pipecolate incorporating enzyme(PIE) of Streptomyces hygroscopicus (34.4%) [9]. AnsC con-tains both sequence motifs associated with adenylate formationand the conserved region LGG-S with the serine residue servingas the covalent attachment point for the 4 0-phosphopantetheinemoiety of peptide synthases [40]. These observations areconsistent with a role of AnsC in the formation of a peptidebond between d-alanine and CHC in the side chain ofansatrienin. No typical epimerization domain is observed forconverting l-alanine to d-alanine. Presumably the carboxylterminus of alanine is activated by adenylation and subsequentlytransferred to the phosphopantetheine arm of AnsC. Peptidebond formation between CHC (activated as a CoA thioester) andthe amino group of the alanine then follows.

The deduced amino acid sequence of NapB is homologous toa hypothetical nonheme oxygenase/halogenase of Amycolatop-sis orientals (32% amino acid identity) and is likely involved inthe modification of naphthomycin. However, naphthomycin C


produced by S. collinus TuÈ 1892 is not halogenated on thenaphthalene ring, differing from naphthomycin A produced byS. collinus TuÈ 105 [14]. Therefore, the role of NapB remainsunclear.

DISCUSSION

In this work two separate sets of AHBA biosynthetic genes havebeen identified in S. collinus DNA. Each of these sets isassociated with genes encoding a type I polyketide synthase,suggesting that they are involved in the biosynthesis of the twoansamycin antibiotics, ansatrienin and naphthomycin, producedby S. collinus. There is no overlap between cosmids carrying thetwo sets of AHBA synthesis genes, indicating that they areseparated by at least 30 kb on the S. collinus genome. Thismakes it very likely that they represent two separate biosyntheticgene clusters, although we cannot categorically rule out thepossibility that both sets of AHBA synthesis genes areassociated with the same PKS, one set upstream and the otherdownstream of the PKS gene. The latter arrangement wouldrequire ansatrienin and naphthomycin to be synthesized on thesame PKS. Synthesis of two different polyketides on the sametype I PKS is precedented in the case of methymycin/pikromycin [41], but seems extremely implausible here inview of the different lengths, 8 vs. 13 chain extension units, andtheir very different processing in ansatrienin compared tonaphthomycin. We therefore conclude that (a) there are separategene clusters involved in the biosynthesis of the ansamycinantibiotics ansatrienin and naphthomycin, and (b) each polyke-tide cluster is independent from the other for AHBA formation.

One of the AHBA biosynthesis gene sets is also associatedwith several genes likely involved in the formation of CHC,including the previously characterized chcA. Because ansatrie-nin A contains a CHC-derived moiety, and disruption of chcAresults in loss of ansatrieinin A production [19], this gene clusteris identified as the one involved in ansatrienin biosynthesis. Thiscluster is the first biosynthetic gene cluster for a benzenoidansamycin to be cloned and partially sequenced. Analysis of anumber of polyketide biosynthetic gene clusters has revealedthat they usually include genes involved in converting primarymetabolites into the precursors required for assembling polyke-tides [8±12]. It is therefore not surprising that one andpotentially more CHC biosynthetic genes are located withinthe ansatrienin biosynthetic gene cluster. This observationsuggests that the chcA gene may be useful as a probe forlocating the gene clusters involved in making other antibioticsthat contain a CHC-derived moiety, such as asukamycin andphospholactomycin [42,43].

The second cluster containing AHBA biosynthetic genesshows striking similarity to the recently characterized rifamycinbiosynthetic gene cluster, both in terms of sequence homologiesand arrangement of the genes [12]. The arrangement of theAHBA biosynthetic genes relative to each other is identical inthe two clusters, and in both clusters they are locatedimmediately downstream from the last module of the PKS andits associated amide synthase. In view of these similarities andthe close structural similarity of naphthomycin and rifamycin,both are naphthalenic ansamycins, it is proposed that this clusteris involved in naphthomycin biosynthesis. As naphthomycinproduction was not detectable in our cultures (K. A. Reynoldsand D. Wilson, unpublished results), we were prevented fromcarrying out an appropriate gene inactivation experiment toconfirm this assignment.

The present work has shown that all the genes identified asinvolved in AHBA biosynthesis in the rifamycin producer,

A. mediterranei, are conserved in each of the two AHBAsynthesis gene sets of S. collinus. The sole exception is theaminoDHQ dehydratase gene, which as in the rifamycin case(rifJ) may be in a distal location in the cluster that has not yetbeen sequenced. This is despite the fact that the function ofsome of these genes is unclear and some may have a regulatoryrole, and that in one of the clusters the arrangement of thesegenes differs greatly from that in the other two clusters. Theirpresence in all three clusters suggests that all these genes areessential for AHBA formation. It is noteworthy that although thelocation and arrangement of all the other AHBA synthase genesis different in the ans cluster than in the nap and rif clusters, theclose association of the AHBA synthase gene (ansF, napF, rifK)with an oxidoreductase gene (ansG, napG, rifL) is the same inall three clusters and in at least one other AHBA synthesis genecluster analyzed (D. Hoffmann, PhD thesis, Bonn, 1997). Thissuggests a functional interaction of some kind between the twogene products.

One of the unique features of the ansatrienin structure is theside chain containing a CHC-derived moiety attached to thepolyketide backbone via a d-alanine [16]. Ansatrienol (myco-trienol) which carries no such side chain has been isolated fromfermentations of S. collinus, suggesting that attachment of theside chain may be a late step in the biosynthetic process [16,44].The peptide synthetase (AnsC) is thought to catalyze at least theformation of the amide bond between the amine group of d-alanine and the carboxylic acid group of CHC (activated as acoenzyme A thioester). As ansatrienin analogs containing 1-cyclohexenylcarbonyl CoA (ansatrienin A4), isobutyryl CoA(ansatrienin A2) and methylbutyryl CoA (ansatrienin A3)-derived moieties, have all been previously identified [2,20] itseems plausible that AnsC can utilize a range of activatedcarboxylic acids. The same group of substrates (isobutyryl CoA,methylbutyryl CoA and cyclohexylcarbonyl CoA) have areutilized by both the loading domain of the avermectin polyketidesynthetase of Streptomyces avermitilis [45], and FabH, whichinitiates fatty acid biosynthesis in Streptomyces glaucescens andpresumably other streptomycetes (v-cyclohexyl fatty acids havebeen previously reported in S. collinus) [46]. The manner inwhich the N-acylated alanine is transferred from AnsC to the 10-OH group of the ansatrienin polyketide backbone is unclear. Forrapamycin biosynthesis in S. hygroscopicus it has beensuggested that RapP is responsible for formation of both apeptide bond between pipecolic acid and the growing rapamycinpolyketide chain (activated as an ACP thioester) and for thesubsequent intramolecular lactonization step [47]. It has beenproposed that this step may be carried out by the RapP motif(HHXXDG) that is conserved in the active sites of chloram-phenicol acetyl transferases and dihydrolipoamide acyltrans-ferases. This motif is not present in AnsC.

The diversity of secondary metabolite biosynthetic pathwaysis thought to have arisen by divergent evolution after geneduplication and by horizontal gene transfer [48]. In this light it isworth noting that the formation of the structurally similaransamycins, naphthomycin and rifamycin, is encoded byclusters which are very similar in the order, orientation andsequences of the AHBA biosynthetic genes. Thus the naphtho-mycin and rifamycin biosynthetic processes appear to be closein an evolutionary sense and have probably arisen throughhorizontal gene transfer processes with little divergent evolution.In contrast the ansatrienin polyketide exhibits significant struc-tural differences to rifamycin and naphthomycin, and differ-ences are clearly also seen in the order, orientation and sequenceof the AHBA biosynthetic genes of the two types of clusters.The ansatrienin and naphthomycin biosynthetic processes


therefore appear to be the result of divergent evolution, despitethe fact that both ansamycin antibiotics are synthesized in thesame organism.

ACKNOWLEDGEMENTS

This work was supported by grants from the National Science Foundation

(MCB-9418581) to K.A.R., the National Institute of Health (AI 20264) to

H.G.F., the NATO [SA, 5-2-05 (CRG-960515)] to H.G.F. and E.L. and the

Deutsche Forschungsgemeinschaft (LE 260/15-1) and Fonds der Che-

mischen Industrie (161537) to E.L. M.B. and R.M. thank the Boehringer

Ingelheim Fonds and the Deutsche Forschungsgemeinschaft, respectively,

for fellowships.

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