title: Engineered cytidine triphosphate synthetase with reduced product inhibition.
abstract: Cytidine triphosphate (CTP) synthetase (CTPS) (EC number 6.3.4.2) is a key enzyme involved in de novo synthesis of CTP. It catalyzes the rate-limiting step of the process due to the product inhibition effects on the enzyme. In this study, a novel CTPS from Corynebacterium glutamicum ATCC 13032 (CgCTPS) was cloned, expressed and characterized. A series of mutagenesis in its N-terminal ammonia ligase (ALase) domain was performed in order to reduce CTP product inhibition. All single mutation variants (D160E, E162A, E168K) lowered product inhibition by lowering the enzyme's binding affinity for CTP. The homology model of CgCTPS showed that D160E mutant caused steric hindrance for the pyrimidine ring of CTP stacking, E162A disrupted the hydrogen bond between CTP ribose and side chain and D168K caused minor localized structure perturbations of CTP binding pocket. The triple mutant of CTPS (D160E-E162A-E168K) with halved Km, doubled Vmax and the 23.5-fold increased IC50 for CTP shows a potential for use in industrial-scale CTP production by its better performance in enzyme kinetics and product inhibition.
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, Nanjing 211816, People's Republic of China.
authors.forename: Mengzhu
authors.initials: M
authors.lastname: Zhu
authors.affiliation: Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Raleigh 27695, USA.
authors.forename: Wujin
authors.initials: W
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, Nanjing 211816, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, Nanjing 211816, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Meng
authors.affiliation: International Cooperation Division, China National Center for Biotechnology Development, Beijing 100036, People's Republic of China.
authors.forename: Dalu
authors.initials: D
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, Nanjing 211816, People's Republic of China Guangzhou Sugarcane Industry Research Institute, Guangzhou 510316, People's Republic of China.
authors.forename: Ting
authors.initials: T
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, Nanjing 211816, People's Republic of China.
authors.forename: Pingkai
authors.initials: P
authors.lastname: Ouyang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, Nanjing 211816, People's Republic of China.
authors.forename: Hanjie
authors.initials: H
authors.lastname: Ying
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, People's Republic of China National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, Nanjing 211816, People's Republic of China Present address: College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, South Puzhu Road 30, Nanjing 211816, People's Republic of China xiej@njtech.edu.cn.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Xie
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D055162:Biocatalysis; D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics; D003570:Cytidine Triphosphate / Q000378:metabolism* / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D008670:Metals; D003570:Cytidine Triphosphate; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase
keywords: Corynebacterium glutamicum ATCC 13032; cytidine triphosphate synthetase; homology modeling; point mutagenesis; product inhibition