Ongoing studies of the biosynthesis of macrolide, polyether, and terpenoid antibiotics will be continued and extended. Among the microbial metabolites to be studied are the macrolides erythromycin (1), methymycin (2), oleandomycin (3) and nargenicin (4); the polyethers monensin (5), lenoremycin (6), and portmicin (7); and the sesquiterpene pentalenolactone (8). Each is of these substances is itself representative of a broad class of related natural products. It is expected that the information gained from studying the biosynthesis of these individual metabolites will be broadly applicable to an understanding of polyketide and terpenoid biosynthesis as well. 1) Based on the use of N-acetylcysteamine (NAC) thioesters as advanced precursors, a systematic study is planned of the polyketide chain-building reactions in the biosynthesis of three closely related macrolide antibiotics erythromycin (1), methymycin (2), and oleandomycin (3) by incorporation specifically labeled, synthetic two-and three-subunit substrates. The biosynthesis of a structurally distinct, but biosynthetically closely related metabolite, nargenicin (4) will also be studied using the same or structurally analogous precursors. These studies are intended to establish the detailed mechanism of chain- elongation in the biosynthesis of reduced polyketides. Following these investigations, studies of the cell-free formation of erythromycin and the 16-membered macrolide, tylosin (9) will be initiated. 2) To test a general stereochemical model of polyether biosynthesis, experiments have been designed to elucidate the details of polyketide chain elongation in the biosynthesis of the polyether ionophoric antibiotics monensin (5), lenoremycin (6), and portmicin (7). Specifically labeled two-, three-, and four-subunit precursors will be fed as the NAC-thioesters in order to test their role in the biosynthesis of polyethers and to demonstrate the intermediacy of olefinic acids in the formation of the reduced polyketide chain. 3) The terpenoid cyclase pentalenene synthetase, an enzyme isolated from Streptomyces UC5319, catalyzes the conversion of farnesyl pyrophosphate to the sesquiterpene pentalenene (10), the parent hydrocarbon of the pentalenolactone family of antibiotics. Using a combination of oligonucleotide and antibody screening, the pentalenene synthetase gene will be cloned and expressed. The availability of cloned DNA will allow sequencing of the synthetase while the increased quantities of cyclase will be used for continued studies of the mechanism and stereochemistry of the cyclization reaction itself.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022172-17
Application #
3270987
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1977-08-01
Project End
1993-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
17
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Brown University
Department
Type
Schools of Arts and Sciences
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
02912
Xie, Xinqiang; Cane, David E (2018) Stereospecific Formation of Z-Trisubstituted Double Bonds by the Successive Action of Ketoreductase and Dehydratase Domains from trans-AT Polyketide Synthases. Biochemistry 57:3126-3129
Xie, Xinqiang; Cane, David E (2018) pH-Rate profiles establish that polyketide synthase dehydratase domains utilize a single-base mechanism. Org Biomol Chem 16:9165-9170
Xie, Xinqiang; Khosla, Chaitan; Cane, David E (2017) Elucidation of the Stereospecificity of C-Methyltransferases from trans-AT Polyketide Synthases. J Am Chem Soc 139:6102-6105
Shah, Dhara D; You, Young-Ok; Cane, David E (2017) Stereospecific Formation of E- and Z-Disubstituted Double Bonds by Dehydratase Domains from Modules 1 and 2 of the Fostriecin Polyketide Synthase. J Am Chem Soc 139:14322-14330
Xie, Xinqiang; Garg, Ashish; Khosla, Chaitan et al. (2017) Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis. J Am Chem Soc 139:3283-3292
Xie, Xinqiang; Garg, Ashish; Khosla, Chaitan et al. (2017) Elucidation of the Cryptic Methyl Group Epimerase Activity of Dehydratase Domains from Modular Polyketide Synthases Using a Tandem Modules Epimerase Assay. J Am Chem Soc 139:9507-9510
Robbins, Thomas; Kapilivsky, Joshuah; Cane, David E et al. (2016) Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase. Biochemistry 55:4476-84
Robbins, Thomas; Liu, Yu-Chen; Cane, David E et al. (2016) Structure and mechanism of assembly line polyketide synthases. Curr Opin Struct Biol 41:10-18
Ostrowski, Matthew P; Cane, David E; Khosla, Chaitan (2016) Recognition of acyl carrier proteins by ketoreductases in assembly line polyketide synthases. J Antibiot (Tokyo) 69:507-10
Xie, Xinqiang; Garg, Ashish; Keatinge-Clay, Adrian T et al. (2016) Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues. Biochemistry 55:1179-86

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