Substrate-bound structures of a ketoreductase from amphotericin modular polyketide synthase

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Modular polyketide synthases (PKSs) are crucial for synthesizing complex polyketide natural products.
• Ketoreductase (KR) domains within PKSs are responsible for controlling the stereochemistry of hydroxyl and methyl substituents at specific carbon positions (C2 and C3) of polyketide intermediates.
• Understanding the molecular mechanisms of KR stereocontrol is essential for engineering PKSs for novel compound synthesis.

Purpose of the Study:

• To elucidate the molecular basis of stereochemical control exerted by Ketoreductase (KR) domains in polyketide synthases (PKSs).
• To investigate the structural and functional roles of the coenzyme (NADP+) and substrate-binding interactions within an A-type KR.
• To determine the influence of the pantetheine handle on the catalytic efficiency and stereochemical outcome of KR reactions.

Main Methods:

• X-ray crystallography was employed to solve the ternary complex structure of a KR from the amphotericin PKS (AmpKR2) bound to NADP+ and a substrate analog.
• Comparative structural analysis was performed using a G355T/Q364H mutant KR complex.
• In vitro functional assays were conducted to assess the impact of the pantetheine handle on enzymatic activity and stereoselectivity.

Main Results:

• The crystal structure of the AmpKR2 ternary complex provides direct evidence for the proposed substrate entry mechanism in A-type KRs, leading to L-hydroxyl substituent formation.
• Structural comparison with a mutant complex revealed insights into the basis of stereospecificity concerning the C2 methyl substituent.
• Functional assays demonstrated that the pantetheine handle significantly influences both the catalytic efficiency and the stereochemical outcome of the KR domain.

Conclusions:

• The solved ternary structure of AmpKR2 offers a detailed molecular understanding of how A-type KRs achieve stereocontrol during polyketide biosynthesis.
• The findings highlight the importance of substrate entry orientation and specific amino acid residues in determining stereochemical outcomes.
• The study underscores the significant role of the pantetheine moiety in modulating KR enzyme function and stereoselectivity, advancing the field of PKS engineering.