Structural and functional analysis of C2-type ketoreductases from modular polyketide synthases

Area of Science:

• Biochemistry
• Structural Biology
• Organic Chemistry

Background:

• Modular polyketide synthases (PKSs) assemble complex molecules but the mechanism for setting α-stereocenters, particularly when reduction is absent, remains unclear.
• Reductase-incompetent ketoreductases (KRs) are hypothesized to function as racemases, facilitating epimerization of polyketide intermediates to achieve the correct stereochemistry in final products like 6-deoxyerythronolide B.

Purpose of the Study:

• To elucidate the structural basis for the proposed racemase activity of reductase-incompetent ketoreductases (KRs) in PKS pathways.
• To investigate the structural differences between reductase-incompetent C2-type KRs and reductase-competent KRs.

Main Methods:

• X-ray crystallography was used to determine the high-resolution (1.88 Å) structure of a C2-type KR from pikromycin synthase.
• Comparative structural analysis was performed between the C2-type KR and known reductase-competent KRs.
• In vivo assays of engineered PKSs were conducted to validate the functional role of C2-type KRs.

Main Results:

• The crystal structure of the C2-type KR revealed distinct features compared to reductase-competent KRs, including an occluded NADPH binding site and increased flexibility in the catalytic tyrosine residue.
• The active-site geometry of C2-type KRs appears optimized for aligning substrate functional groups to facilitate α-proton abstraction.
• In vivo experiments confirmed that C2-type KRs collaborate with specific ketosynthases to control the stereochemistry of α-carbons.

Conclusions:

• The structural and functional data support the role of C2-type KRs as racemases in PKS pathways, crucial for establishing correct stereochemistry in polyketide intermediates.
• The unique structural adaptations of C2-type KRs provide mechanistic insights into stereochemical control during polyketide biosynthesis.
• Understanding these mechanisms can aid in the engineering of PKSs for the synthesis of novel polyketide natural products.