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论文类型：期刊论文

发表时间：2010-04-15

发表刊物：JOURNAL OF BIOTECHNOLOGY

收录刊物：SCIE、EI、PubMed、Scopus

卷号：146

期号：4

页面范围：173-178

ISSN号：0168-1656

关键字：1,3-Propanediol oxidoreductase; Coenzyme specificity; Rational protein design; Computational alanine-scanning mutagenesis; Site-directed mutagenesis; Klebsiella pneumoniae

摘要：1,3-Propanediol has wide applications for large volume markets, particularly in the polymer business. Microbial production of 1,3-propanediol has been considered as a competitor to the traditional petrochemical routes. However, the formation of 1,3-propanediol is limited by the amount of NADH supplied by the oxidative pathway of glycerol dismutation. Previous metabolic flux analysis revealed that relaxation of the coenzyme specificity of 1,3-propanediol oxidoreductase for both NADH and NADPH would increase the production of 1,3-propanediol as well as maintaining the NADH-NAD(+) circle. This work tried to accomplish such a relaxation by rational protein design. Overall binding free energy indicated that the electrostatic energy was the major force discriminating NADH from NADPH. Computational alanine-scanning mutagenesis of the active site residues illustrated that Asp41 was the key residue responsible for the coenzyme specificity. Compared with Asp41Ala, Asp41Gly could further weaken the repulsion between Asp41 and the phosphate group esterified to the 2'-hydroxyl group of the ribose at the adenine end of NADPH. Site-directed mutagenesis was conducted and the relaxation was successfully realized. (C) 2010 Elsevier B.V. All rights reserved.