Biosynthesis of isoprenoids: characterization of a functionally active recombinant 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) from Mycobacterium tuberculosis H37Rv.
Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and Mg(2+). The turnover number of MtIspD is 3.4 s(-1). The Km for MEP and CTP are 43 and 92 muM, respectively. Furthermore, MtIspD shows thermal instable above 50 degrees C. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, P. R. China.
Wenjun
W
Shi
Jianfang
J
Feng
Min
M
Zhang
Xuhui
X
Lai
Shengfeng
S
Xu
Xuelian
X
Zhang
Honghai
H
Wang
D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D019281:Dimerization; D005798:Genes, Bacterial; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013729:Terpenes / Q000378:metabolism*; D013816:Thermodynamics
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013729:Terpenes; C435808:2-C-methyl erythritol 4-phosphate cytidylyltransferase; D009713:Nucleotidyltransferases