Semi-rational engineering of glucosamine-6-phosphate deaminase for catalytic synthesis of glucosamine from D-fructose.

Autor: Zhang ZH; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China., Liao YX; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China., Deng XT; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China., Guan ZB; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China. Electronic address: guanzb@jiangnan.edu.cn.
Jazyk: angličtina
Zdroj: Enzyme and microbial technology [Enzyme Microb Technol] 2025 Feb; Vol. 183, pp. 110552. Date of Electronic Publication: 2024 Nov 27.
DOI: 10.1016/j.enzmictec.2024.110552
Abstrakt: Glucosamine (GlcN), as one of the important derivatives of D-glucose, is formed by the substitution of the hydroxyl group at position 2 of glucose with an amino group. As a bioactive amino monosaccharide, GlcN is known for its various biological effects, including immune enhancement, antioxidant, anti-inflammatory, hepatoprotective, joint pain relief, and alleviation of osteoporosis. These properties highlight the broad applications of GlcN and its derivatives in pharmaceuticals, cosmetics, food production, and other fields, underscoring their promising prospects. Thus, the efficient industrial production of GlcN is gaining increasing attention as well. Here, we report a novel biosynthetic method for GlcN, utilizing engineered Escherichia coli expressing glucosamine-6-phosphate deaminase (GlmD) to directly convert D-fructose into GlcN. The best mutant screened using the Morgan-Elson colorimetric method is the triple mutant G42S/G43C/G136T (designated as GlmD-ZH11), which exhibits approximately 21 times higher catalytic activity towards D-fructose compared to the wild type. Using the purified enzyme of GlmD-ZH11 in shaken flask fermentation for six hours, we achieved a conversion rate of 72.11 % from D-fructose to GlcN. To further elucidate the mechanism behind the enhanced activity of the GlmD-ZH11 mutant, we conducted hydrogen bond network analysis to investigate the hydrogen bond interactions between the mutant and fructose. Additionally, we performed molecular dynamics simulations to study the RMSD and RMSF curves of the mutant. The results indicate that the protein structure of the mutant ZH11 is more stable and binds more tightly to the substrate. Calculations of the solvent-accessible surface area and binding free energy suggested that Thr41, Ser42, Asp72, Gly137, and Ala145 may be key amino acid residues in the catalytic process of ZH11. Finally, based on these findings and the catalytic mechanism of the wild type, we hypothesized a potential catalytic reaction mechanism for the ZH11 mutant.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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