title: Heterologous expression of the methyl carbamate-degrading hydrolase MCD.
abstract: 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.
authors.affiliation: Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan.
authors.forename: Tatheer
authors.initials: T
authors.lastname: Naqvi
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cheesman
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Williams
authors.forename: Peter M
authors.initials: PM
authors.lastname: Campbell
authors.forename: Safia
authors.initials: S
authors.lastname: Ahmed
authors.forename: Robyn J
authors.initials: RJ
authors.lastname: Russell
authors.forename: Colin
authors.initials: C
authors.lastname: Scott
authors.forename: John G
authors.initials: JG
authors.lastname: Oakeshott
mesh_terms: 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
chemical_list: D002219:Carbamates; D008670:Metals; C036868:methyl carbamate; D002265:Carboxylic Ester Hydrolases; C077242:carbamate hydrolase