title: Molecular dynamics studies of modular polyketide synthase ketoreductase stereospecificity.
abstract: Ketoreductases (KRs) from modular polyketide synthases (PKSs) can perform stereospecific catalysis, selecting a polyketide with a D- or L-α-methyl substituent for NADPH-mediated reduction. In this report, molecular dynamics (MD) simulations were performed to investigate the interactions that control stereospecificity. We studied the A1-type KR from the second module of the amphotericin PKS (A1), which is known to be stereospecific for a D-α-methyl-substituted diketide substrate (dkD). MD simulations of two ternary complexes comprised of the enzyme, NADPH, and either the correct substrate, dkD, or its enantiomer (dkL) were performed. The coordinates for the A1/NADPH binary complex were obtained from a crystal structure (PDB entry 3MJS), and substrates were modeled in the binding pocket in conformations appropriate for reduction. Simulations were intended to reproduce the initial weak binding of the polyketide substrate to the enzyme. Long (tens of nanoseconds) MD simulations show that the correct substrate is retained in a conformation closer to the reactive configuration. Many short (up to a nanosecond) MD runs starting from the initial structures display evidence that Q364, three residues N-terminal to the catalytic tyrosine, forms a hydrogen bond to the incorrect dkL substrate to yield an unreactive conformation that is more favorable than the reactive configuration. This interaction is not as strong for dkD, as the D-α-methyl substituent is positioned between the glutamine and the reactive site. This result correlates with experimental findings [Zheng, J., et al. (2010) Structure 18, 913-922] in which a Q364H mutant was observed to lose stereospecificity.
authors.affiliation: †Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States.
authors.forename: Mauro L
authors.initials: ML
authors.lastname: Mugnai
authors.affiliation: ‡Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas 78712, United States.
authors.forename: Yue
authors.initials: Y
authors.lastname: Shi
authors.affiliation: †Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States.|§Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States.
authors.forename: Adrian T
authors.initials: AT
authors.lastname: Keatinge-Clay
authors.affiliation: †Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States.|∥Institute for Computational Engineering and Sciences, The University of Texas at Austin, Austin, Texas 78712, United States.
authors.forename: Ron
authors.initials: R
authors.lastname: Elber
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D003543:Cysteamine / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009249:NADP / Q000737:chemistry; D048630:Polyketide Synthases / Q000737:chemistry*; D013237:Stereoisomerism; D013379:Substrate Specificity; D014631:Valerates / Q000737:chemistry
chemical_list: C000598702:2-methyl-3-oxopentanoate-S-N-acetylcysteamine; D001426:Bacterial Proteins; D014631:Valerates; D009249:NADP; D003543:Cysteamine; D048630:Polyketide Synthases; D000429:Alcohol Oxidoreductases; C087192:polyketide synthase ketoreductase
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