Son, Hyeoncheol Francis Park, Woojin Kim, Sangwoo Kim, Il-Kwon Kim, Kyung-Jin 2024-01-19T08:01:02Z 2024-01-19T08:01:02Z 2023-12-28 2024-01 0141-8130 https://pubs.kist.re.kr/handle/201004/112962 Corynebacterium glutamicum is an industrial workhorse applied in the production of valuable biochemicals. In the process of bio-based chemical production, improving cofactor recycling and mitigating cofactor imbalance are considered major solutions for enhancing the production yield and efficiency. Although, glyceraldehyde-3-phosphate dehydrogenase (GapDH), a glycolytic enzyme, can be a promising candidate for a sufficient NADPH cofactor supply, however, most microorganisms have only NAD-dependent GapDHs. In this study, we performed functional characterization and structure determination of novel NADPH-producing GapDH from C. glutamicum (CgGapX). Based on the crystal structure of CgGapX in complex with NADP cofactor, the unique structural features of CgGapX for NADP stabilization were elucidated. Also, N-terminal additional region (Auxiliary domain, AD) appears to have an effect on enzyme stabilization. In addition, through structure-guided enzyme engineering, we developed a CgGapX variant that exhibited 4.3-fold higher kcat, and 1.2-fold higher kcat/ KM values when compared with wild-type. Furthermore, a bioinformatic analysis of 100 GapX-like enzymes from 97 microorganisms in the KEGG database revealed that the GapX-like enzymes possess a variety of AD, which seem to determine enzyme stability. Our findings are expected to provide valuable information for supplying NADPH cofactor pools in bio-based value-added chemical production. English Elsevier BV Structure-based functional analysis of a novel NADPH-producing glyceraldehyde-3-phosphate dehydrogenase from Corynebacterium glutamicum Article 10.1016/j.ijbiomac.2023.128103 1 International Journal of Biological Macromolecules, v.255 International Journal of Biological Macromolecules 255 N scie scopus 001123959200001 2-s2.0-85177874823 Biochemistry & Molecular Biology Chemistry, Applied Polymer Science Biochemistry & Molecular Biology Chemistry Polymer Science Article ESCHERICHIA-COLI LYSINE PRODUCTION CRYSTAL-STRUCTURE GENE-CLUSTER EXPRESSION SEQUENCE PURIFICATION PATHWAY IDENTIFICATION METABOLISM Corynebacterium glutamicum Unique cofactor selectivity Glyceraldehyde-3-phosphate dehydrogenase NADPH supplying