Structure basis rational engineering of glyceraldehyde-3-phosphate dehydrogenase from Corynebacterium glutamicum for dual NAD/NADP cofactor specificity

Abstract

Abstract Since the discovery of L-glutamate-producing Corynebacterium glutamicum, it has evolved to an industrial workhorse. For bio-based 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 NAD(P)H, is a major prospective solution for the cofactor imbalance issue. In this study, we determined the crystal structure of GapDH from C. glutamicum ATCC13032 (CgGapDH). This structural information was used to construct rational protein engineering strategies. We generated four CgGapDH variants that can produce both NADH and NAPDH. The final CgGapDHL36S/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 CgGapDH variants in complex with NAD(P) permit elucidation of differences between wild-type CgGapDH and variants in relation to cofactor stabilization.