Son, Hyeoncheol Francis Yu, Hyeonjeong Hong, Jiyeon Lee, Donghoon Kim, Il-Kwon Kim, Kyung-Jin 2024-01-19T08:04:10Z 2024-01-19T08:04:10Z 2023-12-28 2023-11 0021-8561 https://pubs.kist.re.kr/handle/201004/113083 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 (CgGAPDH). Based on the structural information, we generated six CgGAPDH variants, CgGAPDH(L36S), CgGAPDH(L36S/T37K), CgGAPDH(L36S/T37K/P192S), CgGAPDH(L36S/T37K/F100V/P192S), CgGAPDH(L36S/T37K/F100L/P192S), and CgGAPDH(L36S/T37K/F100I/P192S), that can produce both NADH and NAPDH. The final CgGAPDH(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 CgGAPDH variants in complex with NAD(P) permit the elucidation of differences between wild-type CgGAPDH and variants in relation to cofactor stabilization. English American Chemical Society Structure-Guided Protein Engineering of Glyceraldehyde-3-phosphate Dehydrogenase from Corynebacterium glutamicum for Dual NAD/NADP Cofactor Specificity Article 10.1021/acs.jafc.3c06176 1 Journal of Agricultural and Food Chemistry, v.71, no.46, pp.17852 - 17859 Journal of Agricultural and Food Chemistry 71 46 17852 17859 N scie scopus 001123357800001 2-s2.0-85178322110 Agriculture, Multidisciplinary Chemistry, Applied Food Science & Technology Agriculture Chemistry Food Science & Technology Article LYSINE PRODUCTION PATHWAY MECHANISM CRYSTAL GENOME glyceraldehyde-3-phosphate cofactor specificity protein engineering cofactor imbalance Corynebacterium glutamicum