A combinatorial library for the binuclear metal center of bacterial phosphotriesterase.
Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three-dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973-7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy-DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4 in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild-type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had < 10(-5) of wild-type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon.
Watkins
L M
LM
Department of Chemistry, Texas A&M University, College Station 77843, USA.
Kuo
J M
JM
Chen-Goodspeed
M
M
Raushel
F M
FM
D043303:Aryldialkylphosphatase; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D004950:Esterases / Q000737:chemistry* / Q000235:genetics; D015723:Gene Library; D007700:Kinetics; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011549:Pseudomonas
D001426:Bacterial Proteins; D008670:Metals; D004950:Esterases; D043303:Aryldialkylphosphatase