title: Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1.
abstract: Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and K(m)  values of the two substrates dUMP and CH(2) H(4) folate were determined to be 15.3 ± 1.25 μM and 0.35 ± 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti-H. pylori inhibitor discovery.
authors.affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Kuifeng
authors.initials: K
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Lili
authors.initials: L
authors.lastname: Chen
authors.forename: Hualiang
authors.initials: H
authors.lastname: Jiang
authors.forename: Xu
authors.initials: X
authors.lastname: Shen
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003856:Deoxyuracil Nucleotides / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013763:Tetrahydrofolates / Q000737:chemistry / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D003856:Deoxyuracil Nucleotides; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D005182:Flavin-Adenine Dinucleotide; C030371:5,6,7,8-tetrahydrofolic acid; D009249:NADP; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase