Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene.
A Klebsiella ozaenae nitrilase which converts the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) to 3,5-dibromo-4-hydroxybenzoic acid has been expressed at 5-10% of the total protein in Escherichia coli from a cloned K. ozaenae DNA segment and purified 10.3-fold to homogeneity. The purified polypeptide is molecular weight 37,000 in size, but the active form of the enzyme is composed of two identical subunits. The purified enzyme exhibits a pH optimum of 9.2 and a temperature optimum of 35 degrees C. The purified enzyme is also quite sensitive to thiol-specific reagents. The nitrilase is highly specific for bromoxynil as substrate with a Km of 0.31 mM and Vmax of 15 mumol of NH3 released/min/mg protein. Analysis of bromoxynil-related substrates indicates the enzyme exhibits preference for compounds containing two meta-positioned halogen atoms. Nucleotide sequence analysis of a 1,212-base pair PstI-HincII DNA segment containing the locus (bxn) encoding the bromoxynil-specific nitrilase reveals a single open reading frame encoding a polypeptide 349 amino acids in length. The predicted sequence of the purified enzyme was derived from the nucleotide sequence of the bxn gene.
Stalker
D M
DM
Calgene Inc., Davis, California 95616.
Malyj
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LD
McBride
K E
KE
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000619:Aminohydrolases / Q000302:isolation & purification*; D001483:Base Sequence; D002874:Chromosome Mapping; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006540:Herbicides / Q000378:metabolism*; D007709:Klebsiella / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
D000596:Amino Acids; D006540:Herbicides; D004262:DNA Restriction Enzymes; D000619:Aminohydrolases; C076915:bromoxynil nitrilase