Engineered Biosynthesis of Novel Polyketides: Dissection of the Catalytic Specificity of the act Ketoreductase

Fu, Huimin; Ebert-Khosla, Susanne; Hopwood, David A.; Khosla, Chaitan

doi:10.1021/ja00089a003

Cited by 124 publications

(119 citation statements)

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“…39,40) Since the aloe plant does not produce the octaketides SEK4/SEK4b and their metabolites and is a rich source of anthrones and anthraquinones (octaketides), it is tempting to speculate that OKS is originally involved in the biosynthesis of anthrones and anthraquinones in the medicinal plant. However, maybe because of misfolding of the heterologously expressed recombinant proteins in E. coli, or because of the absence of interacting proteins of such as yet unidentified ketoreductase, 38) the enzyme possibly yielded SEK4/SEK4b as derailed shunt products just as in the case of the minimal type II PKS (Fig.…”

Section: Novel Pkss From the Aloe Plantmentioning

confidence: 99%

Engineering of Plant Polyketide Biosynthesis

Abe

2008

Chem. Pharm. Bull.

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A growing number of functionally divergent the chalcone synthase (CHS) superfamily type III polyketide synthases (PKSs) have been cloned and characterized, which include recently obtained pentaketide chromone synthase (PCS) and octaketide synthase (OKS) from aloe (Aloe arborescens). Recombinant PCS expressed in Escherichia coli catalyzes iterative condensations of five molecules of malonyl-CoA to produce a pentaketide, 5,7-dihydroxy-2-methylchromone, while OKS carries out sequential condensations of eight molecules of malonyl-CoA to yield aromatic octaketides, SEK4 and SEK4b, the longest polyketides generated by the structurally simple type III PKS. The two enzymes share 91% amino acid sequence identity, maintaining most of the active-site residues of CHS including the Cys-His-Asn catalytic triad. One of the most characteristic features is that the conserved Thr197 of CHS (numbering in Medicago sativa CHS) is uniquely replaced with Met207 in PCS and with Gly207 in OKS, respectively. Site-directed mutagenesis and X-ray crystallographic studies clearly demonstrated that the chemically inert single residue lining the active-site cavity controls the polyketide chain length and the product specificity depending on the steric bulk of the side chain. Finally, on the basis of the crystal structures of both wild-type and M207G-mutant PCS, a triple mutant PCS F80A/Y82A/M207G was constructed and shown to catalyze condensations of nine molecules of malonyl-CoA to produce a novel nonaketide naphthopyrone with a fused tricyclic ring system. Structure-based engineering of the type III PKS superfamily enzymes would thus lead to further production of chemically and structurally divergent unnatural novel polyketides.

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Section: Novel Pkss From the Aloe Plantmentioning

confidence: 99%

Engineering of Plant Polyketide Biosynthesis

Abe

2008

Chem. Pharm. Bull.

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“…Based on these results, the nascent linear decaketide (Fig. 1A) is likely to be the direct substrate for TcmN, which in the absence of any cyclase could slowly undergo aberrant cyclizations as evidenced by the observation of a mixture of cyclized compounds among which only Tcm F2 (18) and SEK15 (21) have been identified (9). To test this hypothesis, we supplemented the cell-free preparation of S. glaucescens WMH1077 (pWHM731) with purified TcmN with the anticipation that TcmN would subvert all aberrant reactions by regiospecifically cyclizing the linear decaketide into Tcm F2.…”

Section: Methodsmentioning

confidence: 95%

Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase.

Shen

Hutchinson

1996

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

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Aromatic polyketides are assembled by a type II (iterative) polyketide synthase (PKS) in bacteria. Understanding the enzymology of such enzymes should provide the information needed for the synthesis of novel polyketides through the genetic engineering of PKSs. Using a previously described cell-free system [B.S. & C.R.H. (1993) Polyketide metabolites are one of the largest groups of natural products, many of which are clinically valuable antibiotics or chemotherapeutic agents, or exhibit other pharmacological activities (1, 2). Despite their apparent structural diversity, they share a common mechanism of biosynthesis. The carbon backbone of a polyketide results from sequential condensation of short fatty acids, such as acetate, propionate, or butyrate, in a manner resembling fatty acid biosynthesis but is catalyzed by polyketide synthases (PKSs). A strong sequence and mechanistic similarity among many of the PKSs has led to two paradigms for explaining polyketide biochemistry: type I PKSs, multifunctional proteins that harbor a distinct active site for every enzyme-catalyzed step, and type II PKSs, multienzyme complexes that carry a single set of iteratively used activities and consist of several largely monofunctional proteins for the synthesis of complex largely reduced polyketides, like macrolide antibiotics, or aromatic polyketides, like tetracyclines (3, 4, 5). Studies of polyketide biosynthesis provide an excellent model for elucidating the structure-function relationship of complex multienzyme systems, and engineered PKSs have considerable potential for synthesizing novel polyketides (6-9).So far, the examination of type II PKS genes in vivo has revealed limited information about how type II PKSs control product structure, in part because the active sites for the RCOSEnz and CH2(CO2H)COSEnz condensation and oligoketide cyclization reactions are used more than once and, therefore, must recognize different substrates in each bondforming event, as exemplified by the tetracenomycin (Tcm) PKS from Streptomyces glaucescens (see Fig. 1) (10-12). We have previously described a cell-free system to facilitate the purification and reconstitution of individual components of type II PKSs (11), using the Tcm PKS as a model. That work showed that the Tcm PKS consists of the TcmJKLMN proteins (10,(12)(13)(14), although components of a S. glaucescens fatty acid synthase may also be required (11,15). We now report the purification of the TcmN polyketide cyclase and its interaction with the other Tcm PKS proteins (i.e., TcmJKLM), which reveals important mechanistic details about how a type II PKS assembles aromatic polyketides. MATERIALS AND METHODSExpression oftcmN in Streptomyces lividans and Purification of TcmN. A 0.5-kb fragment containing the ermE* promoter was subcloned from pWHM862 (16) into the EcoRI/SstI sites of pGEM3zf (Promega) to give pWHM79. The tcmN gene was then moved as a 1.8-kb BglII/SphI fragment from pWHM862 into the BamHI/SphI sites of pWHM79 to give pWHM80, from which a 2.3-kb BamHI/EcoRI fr...

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“…In the absence of cyclases, the KS-CLF and C9 ketoreductase have also been suggested to play key roles in influencing the folding of the polyketide chain (40,53,54). Most minimal PKSs produce C7-C12 cyclized polyketides as major products in the absence of additional tailoring enzymes, indicating that the KS-CLF may promote the regioselectivity of the first cyclization event (55). Analysis of the crystal structure of the actinorhodin (act) KS-CLF showed that buckling of the polyketide chain must occur within the KS-CLF tunnel to accommodate the full-length polyketide chain.…”

Section: Cyclization Of the Polyketide Backbonementioning

confidence: 99%

“…The regioselectivities of the first cyclization steps in 4 and 6 were especially surprising considering that acetateprimed decaketide synthesized by the oxy minimal PKS does indeed cyclize preferentially via C7-C12. For example, the oxy minimal PKS alone produces SEK15 (2a) (33,55), whereas in the presence of act KR, it produces RM20b/c (2b) (44). Therefore, the presence of the amidated starter unit may significantly influence the orientation of the polyketide chain in the active site of the KS-CLF, as well as that of the C9 KR, to result in the unexpected cyclization regioselectivities of 4 and 6.…”

Section: Cyclization Of the Polyketide Backbonementioning

confidence: 99%

Oxytetracycline Biosynthesis

Pickens¹,

Tang

2010

Journal of Biological Chemistry

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Oxytetracycline (OTC) is a broad-spectrum antibiotic that acts by inhibiting protein synthesis in bacteria. It is an important member of the bacterial aromatic polyketide family, which is a structurally diverse class of natural products. OTC is synthesized by a type II polyketide synthase that generates the poly-␤-ketone backbone through successive decarboxylative condensation of malonyl-CoA extender units, followed by modifications by cyclases, oxygenases, transferases, and additional tailoring enzymes. Genetic and biochemical studies have illuminated most of the steps involved in the biosynthesis of OTC, which is detailed here as a representative case study in type II polyketide biosynthesis.

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Engineered Biosynthesis of Novel Polyketides: Dissection of the Catalytic Specificity of the act Ketoreductase

Cited by 124 publications

References 0 publications

Engineering of Plant Polyketide Biosynthesis

Engineering of Plant Polyketide Biosynthesis

Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase.

Oxytetracycline Biosynthesis

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