title: A computational strategy for altering an enzyme in its cofactor preference to NAD(H) and/or NADP(H).
abstract: Coenzyme engineering, especially for altered coenzyme specificity, has been a research hotspot for more than a decade. In the present study, a novel computational strategy that enhances the hydrogen-bond interaction between an enzyme and a coenzyme was developed and utilized to alter the coenzyme preference. This novel computational strategy only required the structure of the target enzyme. No other homologous enzymes were needed to achieve alteration in the coenzyme preference of a certain enzyme. Using our novel strategy, Gox2181 was reconstructed from exhibiting complete NADPH preference to exhibiting dual cofactor specificity for NADH and NADPH. Structure-guided Gox2181 mutants were designed in silico and molecular dynamics simulations were performed to evaluate the strength of hydrogen-bond interactions between the enzyme and the coenzyme NADPH. Three Gox2181 mutants displaying high structure stability and structural compatibility to NADH/NADPH were chosen for experimental confirmation. Among the three Gox2181 mutants, Gox2181-Q20R&D43S showed the highest enzymatic activity by utilizing NADPH as its coenzyme, which was even better than the wild-type enzyme. In addition, isothermal titration calorimetry analysis further verified that Gox2181-Q20R&D43S was able to interact with NADPH but the wild-type enzyme could not. This novel computational strategy represents an insightful approach for altering the cofactor preference of target enzymes.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.
authors.forename: Dongbing
authors.initials: D
authors.lastname: Cui
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.
authors.forename: Lujia
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.
authors.forename: Shuiqin
authors.initials: S
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.
authors.forename: Bei
authors.initials: B
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.
authors.forename: Jinping
authors.initials: J
authors.lastname: Lin
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Y Adam
authors.initials: YA
authors.lastname: Yuan
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism*; D019295:Computational Biology; D017124:Conserved Sequence; D030562:Databases, Protein; D004795:Enzyme Stability; D005103:Expert Systems; D020600:Gluconobacter oxydans / Q000201:enzymology; D006860:Hydrogen Bonding; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D050505:Mutant Proteins; D009243:NAD; D009249:NADP; D010088:Oxidoreductases
keywords: altered coenzyme specificity; molecular dynamics simulation; rational computational design; site-directed mutagenesis; structure stability prediction