Kinetic Analysis of Escherichia coli 2-C-Methyl-d-erythritol-4-phosphate Cytidyltransferase, Wild Type and Mutants, Reveals Roles of Active Site Amino Acids

Abstract

Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (YgbP or IspD) catalyzes the conversion of 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytidine triphosphate (CTP) to 4-diphosphocytidyl-2-C-methylerythritol (CDPME). Pulse chase experiments established that the reaction involves an ordered sequential mechanism with mandatory initial binding of CTP. On the basis of analysis of the previously reported crystal structures of apo-YgbP as well as YgbP complexed with both CTP.Mg(2+) and CDPME.Mg(2+) [Richard, S. B., Bowman, M. E., Kwiatkowski, W., Kang, I., Chow, C., Lillo, A. M., Cane, D. E., and Noel, J. P. (2001) Nat. Struct. Biol. 8, 641-648], a group of active site residues were selected for site-directed mutagenesis and steady-state kinetic analysis. Both Lys27 and Lys213 were shown to be essential to catalytic activity, consistent with their proposed role in stabilization of a pentacoordinate phosphate transition state resulting from in-line attack of the MEP phosphate on the alpha-phosphate of CTP. In addition, Thr140, Arg109, Asp106, and Thr165 were all shown to play critical roles in the binding and proper orientation of the MEP substrate.

Characterization of the Mycobacterium tuberculosis 4-Diphosphocytidyl-2- C -Methyl- d -Erythritol Synthase: Potential for Drug Development

“…The specificity constants (k cat /K m ) of M. tuberculosis IspD for both substrates were lower than those previously determined for E. coli IspD (28), suggesting that IspD of M. tuberculosis is less efficient. However, the K m MEP and K m CTP values used for calculating specificity constants for the E. coli enzyme were 0.37 mM and 0.76 mM, respectively, which are much higher than the K m MEP (3.14 M) and K m CTP (131 M) values determined for the enzyme from the same source, but using a different assay system (31).…”

“…It is assumed that the divalent cations play a similar role in IspD from M. tuberculosis. Not surprisingly, M. tuberculosis IspD showed a high degree of specificity for CTP, which is likely achieved from the pyrimidine base forming hydrogen bonds with the backbone amides of conserved amino acids and stacking interactions inside the catalytic domain (27,28). In fact, mutation of these conserved amino acids abolished or reduced the catalytic activity of E. coli IspD (28).…”

“…Previous enzymatic characterization of IspD orthologs from other organisms were done using 1 H-or 13 C-nuclear magnetic resonance spectroscopy (15,30,31) or thin-layer chromatography-based assays (27,28), which either require expensive equipment or are tedious. In the present study, the PP i released during the reaction catalyzed by IspD was utilized to monitor M. tuberculosis IspD activity.…”

The Non-mevalonate Pathway of Isoprenoid Precursor Biosynthesis

“…MEP reacts with CTP to produce 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME) and release pyrophosphate in the IspD-catalyzed reaction. Of note is the dissection of the EcIspD structure-mechanism relationship by Richard et al (20) based on the combination of informative structures and kinetic analyses (21).…”

Cloning and Characterization of the 2-C-Methyl-D-erythritol 4-Phosphate (MEP) Pathway Genes of a Natural-Rubber Producing Plant,Hevea brasiliensis

“…29) The essential amino acid residues for catalytic activities and substrate binding in the MEP pathway genes of E. coli were highly conserved in H. brasiliensis. [30][31][32][33][34][35][36] In addition, insertion sequences, whose function remains unknown, are highly conserved in plant HDS and HDR. 37) These insertion sequences were also highly conserved in the HDS and HDR of H. brasiliensis.…”