The 6-deoxyerythronolide B synthase (DEBS) is the most extensively studied multimodular polyketide synthase (PKS). During the past project period, we have: (i) solved the X-ray crystal structures of major portions of two DEBS modules;(ii) solved the NMR structure of a prototypical acyl carrier protein domain from DEBS;(iii) investigated mechanistic aspects of key steps in polyketide biosynthesis including intermodular chain transfer, chain elongation, extender unit selection and transfer, ?-ketoreduction, dehydration and macrolactonization;(iv) explored the role of protein-protein interactions in establishing the overall architecture and controlling the selectivity of a PKS module;and (v) interrogated the effect of intramolecularity on the rates of individual reactions within the DEBS catalytic cycle. The biosynthetic engineering implications of these and other insights have also been explored and exploited. During the next project period, we propose to continue our fundamental and applied studies on the structure, mechanism and engineering of DEBS. Specifically, we will attempt to solve the X-ray crystal structures of three new protein constructs including: (i) a large fragment consisting of a nearly complete DEBS module;(ii) a fragment of a DEBS module in which the KS and ACP have been crosslinked to each other;and (iii) a stand-alone acyl transferase (Dsz AT) that is exceptionally efficient at acylating PKS modules in trans. We will also investigate the mechanistic basis for: (i) intra- and inter- modular ketosynthase - acyl carrier protein specificity;(ii) efficient transacylation by Dsz AT;and (iii) rate control of the overall DEBS catalytic cycle, and how it is influenced when unnatural extender units are incorporated. Last but not least, to highlight the practical relevance of the above knowledge, we will: (i) elaborate a newly discovered structure-activity relationship for erythromycin that could lead to explorations within a potentially new binding pocket of the eubacterial ribosome;and (ii) attempt to identify a novel lead substance with medicinally relevant activity against apicomplexan parasites.

Public Health Relevance

The enzymatic assembly line responsible for the biosynthesis of the macrocyclic core of erythromycin is the best-studied example of a multimodular polyketide synthase. This enzyme family produces many important antibiotics in nature. The two principal goals of this project are: (i) to obtain a fundamental understanding of the molecular logic of the erythromycin synthase;and (ii) to exploit this knowledge to make new antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087934-18
Application #
8289626
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Gerratana, Barbara
Project Start
1995-07-05
Project End
2013-08-14
Budget Start
2012-07-01
Budget End
2013-12-30
Support Year
18
Fiscal Year
2012
Total Cost
$356,737
Indirect Cost
$136,214
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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
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
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
Kuo, James; Lynch, Stephen R; Liu, Corey W et al. (2016) Partial In Vitro Reconstitution of an Orphan Polyketide Synthase Associated with Clinical Cases of Nocardiosis. ACS Chem Biol 11:2636-41
Lowry, Brian; Li, Xiuyuan; Robbins, Thomas et al. (2016) A Turnstile Mechanism for the Controlled Growth of Biosynthetic Intermediates on Assembly Line Polyketide Synthases. ACS Cent Sci 2:14-20
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
Klaus, Maja; Ostrowski, Matthew P; Austerjost, Jonas et al. (2016) Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases. J Biol Chem 291:16404-15
Bucher, Cyril; Deans, Richard M; Burns, Noah Z (2015) Highly Selective Synthesis of Halomon, Plocamenone, and Isoplocamenone. J Am Chem Soc 137:12784-7
Khosla, Chaitan (2015) Quo vadis, enzymology? Nat Chem Biol 11:438-41
Lowry, Brian; Walsh, Christopher T; Khosla, Chaitan (2015) In Vitro Reconstitution of Metabolic Pathways: Insights into Nature's Chemical Logic. Synlett 26:1008-1025

Showing the most recent 10 out of 39 publications