Molecular cloning and characterization of a new peptide deformylase from human pathogenic bacterium Helicobacter pylori.
Helicobacter pylori is a gram-negative pathogenic bacterium, which is associated with peptic ulcer disease and gastric cancer. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. In bacteria, peptide deformylase (PDF) catalyzes the removal of a formyl group from the N-termini of nascent polypeptides. Due to its essentiality and absence in mammalian cells, PDF has been considered as an attractive target for the discovery of novel antibiotics. In this work, a new PDF gene (def) from H. pylori strain SS1 was cloned, expressed, and purified in Escherichia coli system. Sequence alignment shows that H. pylori PDF (HpPDF) shares about 40% identity to E. coli PDF (EcPDF). The enzymatic properties of HpPDF demonstrate its relatively high activity toward formyl-Met-Ala-Ser, with K(cat) of 3.4s(-1), K(m) of 1.7 mM, and K(cat) / K(m) of 2000M(-1)s(-1). HpPDF enzyme appears to be fully active at pH between 8.0 and 9.0, and temperature 50 degrees C. The enzyme activity of Co(2+)-containing HpPDF is apparently higher than that of Zn(2+)-containing HpPDF. This present work thereby supplies a potential platform that facilitates the discovery of novel HpPDF inhibitors and further of possible antimicrobial agents against H. pylori.
Han
Cong
C
Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Wang
Qi
Q
Dong
Lei
L
Sun
Haifang
H
Peng
Shuying
S
Chen
Jing
J
Yang
Yiming
Y
Yue
Jianmin
J
Shen
Xu
X
Jiang
Hualiang
H
D000581:Amidohydrolases / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
D001426:Bacterial Proteins; D008670:Metals; D011994:Recombinant Proteins; D000581:Amidohydrolases; C084507:peptide deformylase