title: Identification of UshA as a major enzyme for NAD degradation in Escherichia coli.
abstract: Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH are essential cofactors for many redox biocatalysts. Because these cofactors are consumed in stoichiometric amounts, whole-cell biocatalysts have been routinely employed in order to reduce the costs. To further improve the efficacy of redox biocatalysts, it is essential to maintain the stability of nicotinamide cofactors, for which it is attractive to block degradation pathways for NAD(H). While the biosynthesis of NAD(H) has been well studied, it is less understood how NAD(H) are degraded. Here we demonstrated that UshA was a major periplasmic enzyme for NAD degradation in Escherichia coli. Purified recombinant UshA showed high pyrophosphatase activity with the catalytic efficiencies for hydrolysis of NAD and NADH at 3.7μM(-1)s(-1) and 1.4μM(-1)s(-1), respectively. Deletion of the ushA gene from the chromosome led to faster cell growth and improved extracellular NAD stability by 3-fold under conditions similar to whole-cell biocatalysis. These results significantly enriched our understanding on NAD metabolism, and should facilitate many applications including designing more robust redox biocatalysts.
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China.
authors.forename: Yongjin J
authors.initials: YJ
authors.lastname: Zhou
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China.
authors.forename: Debin
authors.initials: D
authors.lastname: Ji
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors.forename: Xinping
authors.initials: X
authors.lastname: Lin
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors.forename: Yuxue
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors.forename: Yixin
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China.
authors.forename: Wujun
authors.initials: W
authors.lastname: Liu
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China; Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China; The State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China. Electronic address: zhaozb@dicp.ac.cn.
authors.forename: Zongbao K
authors.initials: ZK
authors.lastname: Zhao
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism*; D055162:Biocatalysis; D002463:Cell Membrane Permeability; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D006207:Half-Life; D006868:Hydrolysis; D009243:NAD / Q000378:metabolism*; D009537:Nicotinamide Mononucleotide / Q000378:metabolism; D019897:Periplasm / Q000201:enzymology; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011755:Pyrophosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C406334:ushA protein, E coli; D009243:NAD; D009537:Nicotinamide Mononucleotide; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; C024289:NAD pyrophosphatase
keywords: 5′-Nucleotidase; NAD degradation; Nicotinamide adenine dinucleotide; Pyrophosphatase; UshA; Whole-cell biocatalysis