6-Deoxyerythronolide B Synthase 1 Is Specifically Acylated by a Diketide Intermediate at the β-Ketoacyl-Acyl Carrier Protein Synthase Domain of Module 2

Tsukamoto, Naohiro; Chuck, Jo-Anne; Luo, Guanglin; Kao, Camilla M.; Khosla, Chaitan; Cane, David E.

doi:10.1021/bi961972f

Cited by 26 publications

(18 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Only the 'correct' diastereoisomer was incorporated intact [88]. These results are consistent with recent in vitro studies which demonstrate that the erythromycin diketide NAC intermediate specifically acylates the b-ketosynthase/acyl carrier protein domain of module 2 in DEBS 1 + TE [89].…”

Section: Tetronasinsupporting

confidence: 90%

The Biosynthesis of Aliphatic Polyketides

Staunton

Wilkinson²

1998

Biosynthesis

View full text Add to dashboard Cite

Aliphatic polyketides are a large family of natural products which exhibit an impressive range of biological activities. They are synthesised by a common pathway in which units derived from acetate, propionate and butyrate are condensed onto a growing chain, which is initiated from a range of structurally varied carboxylic acid starter units, in a process closely resembling fatty acid biosynthesis. The intermediates remain bound to the polyketide synthase (PKS) throughout multiple rounds of chain extension, and to a variable degree, reduction of the newly formed b-keto functionality. It is the manner in which each different PKS controls the number and type of extender units added, and the extent and stereochemistry of reduction at each cycle which gives rise to the diverse structural variation seen in this family of natural products. Furthermore, the aglycone product of the PKS may be modified by post-PKS enzymes, including glycosidases, methyltransferases, acylases and oxidative enzymes, to produce still greater diversity. In this article the development of the field will be exemplified by a detailed discussion of the archetypal example of erythromycin A biosynthesis. This will then be followed by further discussion of other relevant areas of research in this field.

show abstract

Section: Tetronasinsupporting

confidence: 90%

The Biosynthesis of Aliphatic Polyketides

Staunton

Wilkinson²

1998

Biosynthesis

View full text Add to dashboard Cite

show abstract

“…Many NAC thioesters of putative polyketide intermediates have successfully been incorporated in vivo (Cane et al, 1993). Acyl groups were directly transferred to the KS from NAC thioesters by what is probably an exchange reaction (Tsukamoto et al, 1996). However, since extender units are transferred by AT domains to ACP domains, it was unclear whether NAC thioesters would be recognized by AT domains in lieu of CoA thioesters.…”

Section: Introductionmentioning

confidence: 99%

Tolerance and Specificity of Recombinant 6-Methylsalicylic Acid Synthase

Richardson

Pohl

Kealey³

et al. 1999

Metabolic Engineering

Self Cite

View full text Add to dashboard Cite

“…We aimed to develop a purification protocol to provide useful quantities of highly purified DEBS 1‐TE and similar PKS multienzymes, free of contaminating enzyme activities. The need for a convenient and efficient method for purifying enzyme in milligram amounts is underlined by recent publications in which only partially purified protein (often 3–4% of total protein) was used for kinetic and specificity studies [20,23,25–29].…”

mentioning

confidence: 99%

Efficient purification and kinetic characterization of a bimodular derivative of the erythromycin polyketide synthase

Bycroft¹,

Weissman²,

Staunton³

et al. 2000

European Journal of Biochemistry

View full text Add to dashboard Cite

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are giant multienzymes that biosynthesize a number of clinically important natural products. The modular nature of PKSs suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides, but the efficacy of such a strategy depends critically on gaining fundamental insight into PKS structure and function, most directly through experiments with purified PKS proteins. Several recent investigations into important aspects of the activity of these enzymes have used only partially purified proteins (often 3±4% of total protein), reflecting how difficult it is to purify these multienzymes in amounts adequate for kinetic and structural analysis. We report here the steady-state kinetic analysis of a typical bimodular PKS, 6-deoxyerythronolide B synthase 1-thioesterase (DEBS 1-TE), purified from recombinant Saccharopolyspora erythraea JCB101 by a new, high-yielding procedure consisting of three steps: ammonium sulfate precipitation, hydrophobic interaction chromatography and size-exclusion chromatography. The method provides 13-fold purification with a recovery of 11% of the applied PKS activity. The essentially homogeneous synthase exhibits an intrinsic methylmalonyl-CoA hydrolase activity, which competes with polyketide chain extension.The most reliable value for the k cat for synthesis of (3S,5R)-dihydroxy-(2R,4R)-dimethyl-n-heptanoic acid-d-lactone is 0.84 min 21, and the apparent K m for (2RS )-methylmalonyl-CoA is 17 mm. This k cat is approximately 10-fold lower than the value reported previously for a differently engineered version of the truncated PKS, DEBS 1+TE. The difference likely reflects the fact that the DEBS 1-TE contains a hybrid acyl carrier protein (ACP) domain in its second module, which lowers its catalytic efficiency.Keywords: polyketide synthase; 6-deoxyerythronolide B synthase 1-thioesterase (DEBS 1-TE); purification; kinetic characterization. Modular polyketide synthases (PKSs) catalyse the biosynthesis of reduced polyketides, a large and structurally diverse class of natural products that exhibit medicinally significant biological properties, including antibiotic, antiparasitic, immunosuppressive and anticancer activities [1]. For example, the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea [1±3] is responsible for biosynthesis of 6-deoxyerythronolide B, the polyketide aglycone of the clinically important antibiotic erythromycin A. The polyketide synthases are giant multienzyme complexes [1±9] that contain a different set or module of enzyme domains to accomplish each successive cycle of chain extension. DEBS contains six such modules housed in three large polypeptides DEBS 1, DEBS 2 and DEBS 3 [10]. In DEBS, as in all modular PKSs, each module determines the structure generated upon chain extension through the choice of unit added and the extent and stereochemistry of b-carbonyl reduction after condensation. Because of the modular nature of these synt...

show abstract

6-Deoxyerythronolide B Synthase 1 Is Specifically Acylated by a Diketide Intermediate at the β-Ketoacyl-Acyl Carrier Protein Synthase Domain of Module 2

Cited by 26 publications

References 25 publications

The Biosynthesis of Aliphatic Polyketides

The Biosynthesis of Aliphatic Polyketides

Tolerance and Specificity of Recombinant 6-Methylsalicylic Acid Synthase

Efficient purification and kinetic characterization of a bimodular derivative of the erythromycin polyketide synthase

Contact Info

Product

Resources

About