Alteration of the Substrate Specificity of a Modular Polyketide Synthase Acyltransferase Domain through Site-Specific Mutations

Reeves, Christopher D.; Murli, Sumati; Ashley, Gary W.; Piagentini, Misty; Hutchinson, C. Richard; McDaniel, Robert

doi:10.1021/bi015864r

Cited by 180 publications

(181 citation statements)

References 25 publications

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“…Given their ability to discriminate between malonyl and methylmalonyl polyketide chain extension units, their substrate specificity has been extensively investigated (26)(27)(28). The previously proposed role of Tyr-742 and Gln-643 in controlling specificity for (2S)-methylmalonyl-CoA (27,28) is clarified by the x-ray structure of the KS-AT homodimer (Fig.…”

Section: Resultsmentioning

confidence: 99%

The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase

Tang

Kim

Mathews

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

266

412

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The x-ray crystal structure of a 194-kDa fragment from module 5 of the 6-deoxyerythronolide B synthase has been solved at 2.7 Å resolution. Each subunit of the homodimeric protein contains a full-length ketosynthase (KS) and acyl transferase (AT) domain as well as three flanking ''linkers.'' The linkers are structurally well defined and contribute extensively to intersubunit or interdomain interactions, frequently by means of multiple highly conserved residues. The crystal structure also reveals that the active site residue Cys-199 of the KS domain is separated from the active site residue Ser-642 of the AT domain by Ϸ80 Å. This distance is too large to be covered simply by alternative positioning of a statically anchored, fully extended phosphopantetheine arm of the acyl carrier protein domain from module 5. Thus, substantial domain reorganization appears necessary for the acyl carrier protein to interact successively with both the AT and the KS domains of this prototypical polyketide synthase module. The 2.7-Å KS-AT structure is fully consistent with a recently reported lower resolution, 4.5-Å model of fatty acid synthase stucture, and emphasizes the close biochemical and structural similarity between polyketide synthase and fatty acid synthase enzymology. modular megasynthase ͉ multienzyme assembly ͉ polyketide synthase

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Section: Resultsmentioning

confidence: 99%

The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase

Tang

Kim

Mathews

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

266

412

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“…, and His 506 ) were characterized (1,42). However, two other highly crucial conserved residues vary for OzmM-AT1: Ala 80 in place of His and Arg 106 in place of Gln.…”

mentioning

confidence: 99%

Oxazolomycin Biosynthesis in Streptomyces albus JA3453 Featuring an “Acyltransferase-less” Type I Polyketide Synthase That Incorporates Two Distinct Extender Units

Zhao

Coughlin

et al. 2010

Journal of Biological Chemistry

115

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The oxazolomycins (OZMs) are a growing family of antibiotics produced by several Streptomyces species that show diverse and important antibacterial, antitumor, and anti-human immunodeficiency virus activity. Oxazolomycin A is a peptidepolyketide hybrid compound containing a unique spiro-linked ␤-lactone/␥-lactam, a 5-substituted oxazole ring. The oxazolomycin biosynthetic gene cluster (ozm) was identified from Streptomyces albus JA3453 and localized to 79.5-kb DNA, consisting of 20 open reading frames that encode non-ribosomal peptide synthases, polyketide synthases (PKSs), hybrid nonribosomal peptide synthase-PKS, trans-acyltransferases (trans-ATs), enzymes for methoxymalonyl-acyl carrier protein (ACP) synthesis, putative resistance genes, and hypothetical regulation genes. In contrast to classical type I polyketide or fatty acid biosynthases, all 10 PKS modules in the gene cluster lack cognate ATs. Instead, discrete ATs OzmM (with tandem domains OzmM-AT1 and OzmM-AT2) and OzmC were equipped to carry out all of the loading functions of both malonyl-CoA and methoxymalonyl-ACP extender units. Strikingly, only OzmM-AT2 is required for OzmM activity for OZM biosynthesis, whereas OzmM-AT1 seemed to be a cryptic AT domain. The above findings, together with previous results using isotope-labeled precursor feeding assays, are assembled for the OZM biosynthesis model to be proposed. The incorporation of both malonylCoA (by OzmM-AT2) and methoxymalonyl-ACP (by OzmC) extender units seemed to be unprecedented for this class of trans-AT type I PKSs, which might be fruitfully manipulated to create structurally diverse novel compounds.

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“…Condensation 10 of the biosynthesis of the concanamycin aglycone would appear to require ethylmalonyl-CoA. The examination of the AT domain for this extension cycle is consistent with a preference for ethylmalonyl-CoA over methylmalonyl-CoA (Reeves et al, 2001) (see also below). ORFs 18* and 19* appear to encode a crotonyl-CoA reductase and bhydroxybutyryl-CoA reductase, respectively, which could both play a role in furnishing butyrate extender units.…”

Section: Provision Of Extender Units and Modification Of The Pksmentioning

confidence: 59%

“…Examination of the encoded AT domains of CON polypeptides for distinctive amino acid sequence motifs allows the chemical nature of the extender unit recruited by each AT domain (especially malonate versus methylmalonate or ethylmalonate) to be inferred (Haydock et al, 1995;Lau et al, 1999;Reeves et al, 2001;Del Vecchio et al, 2003). A signature motif for AT domains that incorporate methoxymalonate has, because of the rarity of this extension unit, hitherto eluded definition (but see below).…”

Section: Organization Of the Concanamycin Pksmentioning

confidence: 99%

Organization of the biosynthetic gene cluster for the macrolide concanamycin A in Streptomyces neyagawaensis ATCC 27449

et al. 2005

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The macrolide antibiotic concanamycin A has been identified as an exceptionally potent inhibitor of the vacuolar (V-type) ATPase. Such compounds have been mooted as the basis of a potential drug treatment for osteoporosis, since the V-ATPase is involved in the osteoclast-mediated bone resorption that underlies this common condition. To enable combinatorial engineering of altered concanamycins, the biosynthetic gene cluster governing the biosynthesis of concanamycin A has been cloned from Streptomyces neyagawaensis and shown to span a region of over 100 kbp of contiguous DNA. An efficient transformation system has been developed for S. neyagawaensis and used to demonstrate the role of the cloned locus in the formation of concanamycin A. Sequence analysis of the 28 ORFs in the region has revealed key features of the biosynthetic pathway, in particular the biosynthetic origin of portions of the backbone, which arise from the unusual polyketide building blocks ethylmalonyl-CoA and methoxymalonyl-ACP, and the origin of the pendant deoxysugar moiety 49-O-carbamoyl-29-deoxyrhamnose, as well as the presence of a modular polyketide synthase (PKS) encoded by six giant ORFs. Examination of the methoxymalonyl-specific acyltransferase (AT) domains has led to recognition of an amino acid sequence motif which can be used to distinguish methylmalonyl-CoA-from methoxymalonyl-ACP-specific AT domains in natural PKSs. INTRODUCTIONPolyketide macrolides constitute a large family of bioactive natural products, including many compounds with important clinical applications in both human and veterinary medicine. Concanamycin A (folimycin) is an unusual macrolide, first isolated from Streptomyces diastatochromogenes S-45 (Kinashi et al., 1984), whose absolute configuration was established as shown in Fig. 1 (Westley et al., 1984). Concanamycin A and closely related compounds (concanamycins B-F) are characterized by an 18-membered tetraenic macrolide ring incorporating a methyl enol ether, and by a b-hydroxyhemiacetal side chain. They are closely related to the 16-membered macrolides called bafilomycins (Goetz et al., 1985) and also share structural features with the antifungal macrodiolide, elaiophylin A (Arcamone et al., 1959) whose biosynthetic pathway we recently elucidated (Haydock et al., 2004). These unusual macrolides have been dubbed the 'plecomacrolides' (from the Greek word plectos, describing their unusually folded side chain) (Bindseil & Zeeck, 1994).The concanamycins exhibit a wide range of important biological activities: antiviral (Omura et al., 1983), antiprotozoal (Omura et al., 1983 and antineoplastic (SekiAsano et al., 1994). These activities are thought to be due, at least in part, to their potent inhibition of the vacuolar (Vtype) H + -ATPase (Muroi et al., 1993) mediated by their binding to the membrane-spanning V 0 component of the ATPase (Drose et al., 2001). Interest in these compounds has increased recently with the recognition of the importance of the V-type ATPase in osteoclast-mediated resorption of bone (Lait...

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Alteration of the Substrate Specificity of a Modular Polyketide Synthase Acyltransferase Domain through Site-Specific Mutations

Cited by 180 publications

References 25 publications

The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase

The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase

Oxazolomycin Biosynthesis in Streptomyces albus JA3453 Featuring an “Acyltransferase-less” Type I Polyketide Synthase That Incorporates Two Distinct Extender Units

Organization of the biosynthetic gene cluster for the macrolide concanamycin A in Streptomyces neyagawaensis ATCC 27449

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