High-resolution structures of formate dehydrogenase from Candida boidinii

Area of Science:

• Biocatalysis and Enzyme Engineering
• Structural Biology
• Biotechnology

Background:

• Enzymatic recovery of nicotinamide adenine dinucleotide (NADH) is crucial for biotechnological processes like asymmetric hydrogenation.
• NAD(+)-dependent formate dehydrogenase (FDH) from yeast Candida boidinii (CbFDH) is widely used but has limitations in stability and activity.
• Previous enzyme engineering efforts focused on improving CbFDH, but lacked detailed structural insights.

Purpose of the Study:

• To clarify the enzymatic mechanism of CbFDH using high-resolution X-ray structural studies.
• To enhance the effectiveness and operational stability of CbFDH as a cofactor regenerator.
• To explain biochemical differences between yeast and bacterial FDH enzymes.

Main Methods:

• Designed two single-point mutants of CbFDH using a surface engineering approach.
• Obtained protein crystals suitable for high-resolution X-ray structural analysis.
• Determined crystal structures of the engineered CbFDH mutants.

Main Results:

• Mutations significantly improved the crystallizability of CbFDH.
• The engineered mutants exhibited enhanced catalytic properties and improved enzyme stability.
• Crystal structures provided insights into enzymatic mechanisms and differences between yeast and bacterial FDH.

Conclusions:

• The designed mutations provide a basis for further rational mutagenesis studies.
• Structural information from engineered CbFDH aids in developing more effective enzymatic cofactor regeneration systems.
• This work advances the application of biocatalysis in enantioselective synthesis.