Kinetic analysis of Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase, wild type and mutants, reveals roles of active site amino acids.
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.
Richard
Stéphane B
SB
Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.
Lillo
Antonietta M
AM
Tetzlaff
Charles N
CN
Bowman
Marianne E
ME
Noel
Joseph P
JP
Cane
David E
DE
D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002457:Cell Extracts; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D002457:Cell Extracts; D011994:Recombinant Proteins; C435808:2-C-methyl erythritol 4-phosphate cytidylyltransferase; D009713:Nucleotidyltransferases