Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity.
2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4Å resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India.
Sagar
S
Chittori
H S
HS
Savithri
M R N
MR
Murthy
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism; D002951:Citrates / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods*; D005232:Fatty Acids, Volatile / Q000378:metabolism; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012486:Salmonella typhimurium / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002951:Citrates; D005232:Fatty Acids, Volatile; C031605:2-methylcitric acid; D002950:Citrate (si)-Synthase; C112399:2-methylcitrate synthase; D007652:Oxo-Acid-Lyases