Complete pyridine-nucleotide-specific conversion of an NADH-dependent ferredoxin reductase

Area of Science:

• Biochemistry
• Enzyme Engineering
• Metabolic Engineering

Background:

• Enzyme coenzyme specificity is crucial for engineered biosynthesis.
• Photosynthetic organisms naturally produce abundant NADPH, making it ideal for carbon-neutral production.
• Converting NADH-specific enzymes to NADPH-specific ones facilitates efficient biosynthesis in these organisms.

Purpose of the Study:

• To engineer an NADH-specific ferredoxin reductase (BphA4) into an NADPH-dependent enzyme.
• To enable efficient carbon-neutral production of biological compounds in photosynthetic organisms.

Main Methods:

• Employed structure-based systematic mutations and site-directed random mutagenesis.
• Focused on altering the NADH-recognition loop of BphA4.
• Utilized a semi-empirical prediction method to assess mutation effects.

Main Results:

• Developed a CRG mutant (Cys175-Arg176-Gly177) from wild-type BphA4.
• The CRG mutant exhibited high specificity and activity for NADPH.
• Biochemical and structural properties of the CRG mutant for NADPH mirrored the wild-type's for NADH.
• Prediction methods indicated the CRG mutant as a top-performing NADPH-specific variant.

Conclusions:

• Successfully converted an NADH-specific ferredoxin reductase to an NADPH-dependent form.
• The engineered enzyme maintains desirable biochemical and structural properties.
• This strategy is promising for enhancing carbon-neutral biosynthesis in photosynthetic systems.