Evidence that the methylesterase of bacterial chemotaxis may be a serine hydrolase.
CheB, the methylesterase of chemotactic bacteria, catalyzes the hydrolysis of glutamyl-methyl esters in bacterial chemoreceptor proteins. The two cysteines predicted by the amino acid sequence of CheB were replaced by alanine residues. The resulting mutants, Cys207-Ala, Cys309-Ala and a double cysteine mutant Cys207-Ala/Cys309-Ala, retained methylesterase activity, indicating that sulfhydryls are not crucial for CheB mediated catalysis. A homology search revealed a conserved serine active-site region between residues 162 and 166 which is homologous to the active-site region of acetylcholine esterases, suggesting that Ser164 of CheB is the active-site nucleophile. Oligonucleotide-directed mutagenesis was used to change the serine to a cysteine. This Ser164-Cys mutant had less than 2% of the wild-type activity. Unlike the serine proteinases which utilize a 'catalytic triad' mechanism, CheB does not have the conserved histidine and aspartic acid residues located in positions N-terminal to the active-site serine. In addition, CheB is not labeled with di-isopropylfluorophosphate, a potent inhibitor of other serine hydrolases. A novel mechanism is proposed for CheB involving substrate-assisted catalysis to account for these apparent anomalies.
Krueger
J K
JK
Department of Chemistry, Princeton University, NJ 08544.
Stock
J
J
Schutt
C E
CE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002630:Chemotactic Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002633:Chemotaxis; D004926:Escherichia coli / Q000235:genetics; D007531:Isoflurophate / Q000378:metabolism; D008969:Molecular Sequence Data; D012694:Serine / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*
D001426:Bacterial Proteins; D002630:Chemotactic Factors; D007531:Isoflurophate; C069656:CheB protein, Bacteria; D012694:Serine; D002265:Carboxylic Ester Hydrolases; C041144:chemotactic protein methylesterase; D012697:Serine Endopeptidases