Structure-Guided Protein Engineering of Glyceraldehyde-3-phosphate Dehydrogenase from Corynebacterium glutamicum for Dual NAD/NADP Cofactor Specificity.

Since the discovery of l-glutamate-producing Corynebacterium glutamicum , it has evolved to be an industrial workhorse. For biobased chemical production, suppling sufficient amounts of the NADPH cofactor is crucial. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme that converts glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate and produces NADH, is a major prospective solution for the cofactor imbalance issue. In this study, we determined the crystal structure of GAPDH from C. glutamicum ATCC13032 ( Cg GAPDH). Based on the structural information, we generated six Cg GAPDH variants, Cg GAPDH L36S , Cg GAPDH L36S/T37K , Cg GAPDH L36S/T37K/P192S , Cg GAPDH L36S/T37K/F100V/P192S , Cg GAPDH L36S/T37K/F100L/P192S , and Cg GAPDH L36S/T37K/F100I/P192S , that can produce both NADH and NAPDH. The final Cg GAPDH L36S/T37K/F100V/P192S variant showed a 212-fold increase in enzyme activity for NADP as well as 200% and 30% increased activity for the G3P substrate under NAD and NADP cofactor conditions, respectively. In addition, crystal structures of Cg GAPDH variants in complex with NAD(P) permit the elucidation of differences between wild-type Cg GAPDH and variants in relation to cofactor stabilization.