Structural and Stereochemical Analysis of a Modular Polyketide Synthase Ketoreductase Domain Required for the Generation of a cis-Alkene

• Published in: Chemistry & biology Vol. 20; no. 6; pp. 772 - 783
• Main Authors: Bonnett, Shilah A., Whicher, Jonathan R., Papireddy, Kancharla, Florova, Galina, Smith, Janet L., Reynolds, Kevin A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 20-06-2013
• Subjects: Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - metabolism; Alkenes - chemistry; Alkenes - metabolism; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding Sites; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Kinetics; Molecular Sequence Data; Protein Structure, Tertiary; Sequence Alignment; Stereoisomerism; Substrate Specificity
• ISSN: 1074-5521; 1879-1301
• DOI: 10.1016/j.chembiol.2013.04.014

The formation of an activated cis-3-cyclohexylpropenoic acid by Plm1, the first extension module of the phoslactomycin polyketide synthase, is proposed to occur through an L-3-hydroxyacyl-intermediate as a result of ketoreduction by an A-type ketoreductase (KR). Here, we demonstrate that the KR domain of Plm1 (PlmKR1) catalyzes the formation of an L-3-hydroxyacyl product. The crystal structure of PlmKR1 revealed a well-ordered active site with a nearby Trp residue characteristic of A-type KRs. Structural comparison of PlmKR1 with B-type KRs that produce D-3-hydroxyacyl intermediates revealed significant differences. The active site of cofactor-bound A-type KRs is in a catalysis-ready state, whereas cofactor-bound B-type KRs are in a precatalytic state. Furthermore, the closed lid loop in substrate-bound A-type KRs restricts active site access from all but one direction, which is proposed to control the stereochemistry of ketoreduction.
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•PlmKR1 is an A-type KR that catalyzes the formation of an L-configured alcohol•Structure analysis reveals a cofactor-assisted formation of a catalysis-ready state•Conformation of the lid loop controls substrate access to the active site
Bonnett et al. demonstrate that the ketoreductase (KR) domain of Plm1 (PlmKR1), from the phoslactomycin PKS, is an A-type KR that catalyzes the stereospecific reduction of the β-keto intermediate to generate an L-configured alcohol. Stereochemistry of ketoreduction is dictated by the conformation of the lid loop.