TY - JOUR
T1 - Evidence that the methylesterase of bacterial chemotaxis may be a serine hydrolase
AU - Krueger, Joanna K.
AU - Stock, J.
AU - Schutt, C. E.
N1 - Funding Information:
We thank Dr. Jim Mottonen for his enlightenment regardingt he sequenceh omologyo f CheB with acetylcholine esterasesa nd Dr. Michael Rozyski for his invaluablea dvice and suggestionsW. e also thank Dr. Robert Gerard (University of Texas, Southwestern Medical School) for the use of his mutagenesifsa cili-ties and expertise. Thisr esearch was supportedb y grants from the National Institute of Health.
Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 1992/3/12
Y1 - 1992/3/12
N2 - 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.
AB - 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.
KW - Active-site serine
KW - Chemotaxis
KW - Di-isopropylfluorophosphate
KW - Methylesterase
KW - Substrate-assisted catalysis
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U2 - 10.1016/0167-4838(92)90220-8
DO - 10.1016/0167-4838(92)90220-8
M3 - Article
C2 - 1547277
AN - SCOPUS:0026508060
SN - 0167-4838
VL - 1119
SP - 322
EP - 326
JO - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
JF - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
IS - 3
ER -