Mutation of Gly51 to serine in the P-loop of Lactobacillus casei folylpolyglutamate synthetase abolishes activity by altering the conformation of two adjacent loops.
Based upon the three-dimensional structure of Lactobacillus casei folylpolyglutamate synthetase (FPGS), site-directed mutagenesis studies were performed on three residues associated with the ATPase site: Gly51, Ser52 and Ser73. Gly51 and Ser52 are at the end of the P-loop, which is involved in triphosphate binding. A G51S mutant enzyme and a G51S/S52T double-mutant enzyme were made in order to alter the FPGS P-loop to more closely resemble the sequences found in other ATPase and GTPase enzymes. Ser73 is on a neighboring loop (the Omega-loop) and precedes a proline residue found to be in a cis conformation. The carbonyl O atom of Ser73 is one of the protein ligands for the essential Mg(2+) ion involved in ATP binding and hydrolysis and the Omega-loop is involved in binding the folate substrate 5,10-methylenetetrahydrofolate. The serine residue was mutated to alanine and this is the only one of the three mutants which retains some FPGS activity. The structures of the G51S, G51S/S52T and S73A mutant proteins have been solved to high resolution, along with the structure of the apo wild-type FPGS. The P-loop in both the G51S and G51S/S52T mutant proteins remains unaltered, yet both structures show a large conformational rearrangement of the Omega-loop in which a cis-Pro residue has switched conformation to a trans-peptide. The structure of the Omega-loop is severely disrupted and as a consequence structural rearrangements are observed in the peptide linker joining the two domains of the enzyme. Magnesium binding in the active site is also disrupted by the presence of the serine side chain at position 51 and by the repositioning of the carbonyl O atom of Ser73 and a water molecule is bound in place of the Mg(2+) ion. The S73A mutant protein retains the cis-Pro configuration in the Omega-loop and the Mg(2+) site remains intact. The cis-Pro is also observed in the structure of the substrate-free form of FPGS (apoFPGS), maintained in the absence of Mg(2+) by a hydrogen-bonding network involving water molecules in the active site. It is only in the complete absence of water or Mg(2+) in the binding site that the cis-Pro switches to the trans conformation.
Smith
Clyde A
CA
Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA, USA. csmith@slac.stanford.edu
Cross
Jennifer A
JA
Bognar
Andrew L
AL
Sun
Xiaolin
X
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics; D011392:Proline / Q000737:chemistry; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry* / Q000235:genetics
D001051:Apoenzymes; D001426:Bacterial Proteins; D012694:Serine; D011392:Proline; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008274:Magnesium; D005998:Glycine