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.
Corynebacterium glutamicum
(
C. glutamicum
) has been considered a very important and meaningful industrial microorganism for the production of amino acids worldwide. To produce amino acids, cells require nicotinamide adenine dinucleotide phosphate (NADPH), which is a biological reducing agent. The pentose phosphate pathway (PPP) can supply NADPH in cells via the 6-phosphogluconate dehydrogenase (6PGD) enzyme, which is an oxidoreductase that converts 6-phosphogluconate (6PG) to ribulose 5-phosphate (Ru5P), to produce NADPH. In this study, we identified the crystal structure of 6PGD_apo and 6PGD_NADP from
C. glutamicum
ATCC 13032 (
Cg
6PGD) and reported our biological research based on this structure. We identified the substrate binding site and co-factor binding site of
Cg
6PGD, which are crucial for understanding this enzyme. Based on the findings of our research,
Cg
6PGD is expected to be used as a NADPH resource in the food industry and as a drug target in the pharmaceutical industry.
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