TY - JOUR
T1 - Biosynthesis of isoprenoids
T2 - Characterization of a functionally active recombinant 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) from Mycobacterium tuberculosis H37Rv
AU - Shi, Wenjun
AU - Feng, Jianfang
AU - Zhang, Min
AU - Lai, Xuhui
AU - Xu, Shengfeng
AU - Zhang, Xuelian
AU - Wang, Honghai
PY - 2007/11
Y1 - 2007/11
N2 - 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 μM, respectively. Furthermore, MtIspD shows thermal instable above 50°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.
AB - 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 μM, respectively. Furthermore, MtIspD shows thermal instable above 50°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.
KW - Circular dichroism
KW - Enzyme
KW - MEP cytidyltransferase
KW - MEP pathway
KW - Mycobacterium tuberculosis
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U2 - 10.5483/bmbrep.2007.40.6.911
DO - 10.5483/bmbrep.2007.40.6.911
M3 - Article
C2 - 18047786
AN - SCOPUS:36949007177
SN - 1225-8687
VL - 40
SP - 911
EP - 920
JO - Journal of Biochemistry and Molecular Biology
JF - Journal of Biochemistry and Molecular Biology
IS - 6
ER -