Heterologous expression of the methyl carbamate-degrading hydrolase MCD.
The methyl carbamate-degrading hydrolase (MCD) of Achromobacter WM111 has considerable potential as a pesticide bioremediation agent. However this potential has been unrealisable until now because of an inability to express MCD in heterologous hosts such as Escherichia coli. Herein, we describe the first successful attempt to express appreciable quantities of MCD in active form in E. coli, and the subsequent characterisation of the heterologously expressed material. We find that the properties of this material closely match the previously reported properties of MCD produced from Achromobacter WM111. This includes the presence of two distinct forms of the enzyme that we show are most likely due to the presence of two functional translational start sites. The purified enzyme catalyses the hydrolysis of a carbamate (carbaryl), a carboxyl ester (alpha-naphthyl acetate) and a phophotriester (dimethyl umbelliferyl phosphate) and it is relatively resistant to thermal and solvent-mediated denaturation. The robust nature and catalytic promiscuity of MCD suggest that it could be exploited for various biotechnological applications.
Naqvi
Tatheer
T
Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan.
Cheesman
Matthew J
MJ
Williams
Michelle R
MR
Campbell
Peter M
PM
Ahmed
Safia
S
Russell
Robyn J
RJ
Scott
Colin
C
Oakeshott
John G
JG
D042402:Achromobacter / Q000201:enzymology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002219:Carbamates / Q000737:chemistry / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013696:Temperature
D002219:Carbamates; D008670:Metals; C036868:methyl carbamate; D002265:Carboxylic Ester Hydrolases; C077242:carbamate hydrolase