Improving Cofactor Promiscuity of HMG-CoA Reductase from Ruegeria pomeroyi Through Rational Design

Area of Science:

• Biochemistry
• Enzyme Engineering
• Metabolic Engineering

Background:

• The mevalonate pathway is essential for producing isopentenyl pyrophosphate (IPP), the precursor to all terpenoids.
• 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the rate-limiting enzyme in this pathway, catalyzing HMG-CoA to mevalonate reduction using NAD(P)H.
• Enhancing HMGR's cofactor promiscuity can overcome limitations in substrate utilization and boost terpenoid biosynthesis.

Purpose of the Study:

• To heterologously express and characterize rpHMGR from Ruegeria pomeroyi in Escherichia coli.
• To engineer HMGR for dual cofactor utilization (NADH and NADPH) through rational design.
• To assess the stability and catalytic activity of the engineered HMGR mutant.

Main Methods:

• Heterologous expression of rpHMGR in Escherichia coli BL21(DE3).
• Molecular Operating Environment (MOE)-assisted rational design to engineer the cofactor binding site.
• Biochemical assays to determine enzyme activity, cofactor preference, and pH stability of the wild-type and mutant HMGR.

Main Results:

• rpHMGR predominantly utilizes NADH, with limited NADPH activity.
• A D154K mutant was successfully engineered, showing a 53.7-fold increase in NADPH activity.
• The D154K mutant maintained high catalytic activity across a broad pH range (6-8) with both NADH and NADPH, without compromising stability.

Conclusions:

• Rational design effectively engineered HMGR for dual cofactor utilization, significantly enhancing NADPH activity.
• The D154K mutant offers broader cofactor flexibility, crucial for optimizing terpenoid production.
• This study advances the understanding of HMGR-cofactor interactions and provides a basis for further enzyme engineering efforts.