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
Khosla, Chaitan; Herschlag, Daniel; Cane, David E et al. (2014) Assembly line polyketide synthases: mechanistic insights and unsolved problems. Biochemistry 53:2875-83
Edwards, Andrea L; Matsui, Tsutomu; Weiss, Thomas M et al. (2014) Architectures of whole-module and bimodular proteins from the 6-deoxyerythronolide B synthase. J Mol Biol 426:2229-45
O'Brien, Robert V; Davis, Ronald W; Khosla, Chaitan et al. (2014) Computational identification and analysis of orphan assembly-line polyketide synthases. J Antibiot (Tokyo) 67:89-97
Dunn, Briana J; Watts, Katharine R; Robbins, Thomas et al. (2014) Comparative analysis of the substrate specificity of trans- versus cis-acyltransferases of assembly line polyketide synthases. Biochemistry 53:3796-806
Garg, Ashish; Xie, Xinqiang; Keatinge-Clay, Adrian et al. (2014) Elucidation of the cryptic epimerase activity of redox-inactive ketoreductase domains from modular polyketide synthases by tandem equilibrium isotope exchange. J Am Chem Soc 136:10190-3
Kuo, James; Khosla, Chaitan (2014) The initiation ketosynthase (FabH) is the sole rate-limiting enzyme of the fatty acid synthase of Synechococcus sp. PCC 7002. Metab Eng 22:53-9
Walker, Mark C; Thuronyi, Benjamin W; Charkoudian, Louise K et al. (2013) Expanding the fluorine chemistry of living systems using engineered polyketide synthase pathways. Science 341:1089-94
Dunn, Briana J; Khosla, Chaitan (2013) Engineering the acyltransferase substrate specificity of assembly line polyketide synthases. J R Soc Interface 10:20130297
You, Young-Ok; Khosla, Chaitan; Cane, David E (2013) Stereochemistry of reductions catalyzed by methyl-epimerizing ketoreductase domains of polyketide synthases. J Am Chem Soc 135:7406-9
Xiao, Xirui; Yu, Xingye; Khosla, Chaitan (2013) Metabolic flux between unsaturated and saturated fatty acids is controlled by the FabA:FabB ratio in the fully reconstituted fatty acid biosynthetic pathway of Escherichia coli. Biochemistry 52:8304-12

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