Characterization of the phenylurea hydrolases A and B: founding members of a novel amidohydrolase subgroup.
Mycobacterium brisbanense strain JK1, a bacterium capable of degrading the herbicide diuron, was isolated from herbicide-exposed soil. A gene/enzyme system with diuron hydrolase activity was isolated from this strain and named PUH (phenylurea hydrolase) B (puhB/PuhB) because of its close similarity to the previously characterized PUH A (puhA/PuhA). Both PUHs were heterologously expressed, purified and characterized. The PUHs were found to oligomerize as hexamers in solution, with each monomer containing a mononuclear Zn2+ active site. Sequence analysis showed that these enzymes belong to the metal-dependent amidohydrolase superfamily, although they contain a hitherto unreported Asn-X-His metal-binding motif and appear to form a novel sub-group within this superfamily. The effects of temperature and solvent on the enzymes were characterized. Determination of the kinetic parameters of the PUHs was used alongside Brønsted plots to develop a plausible catalytic mechanism, which is similar to that used by urease. In addition to the primary PUH activity, both enzymes are catalytically promiscuous, efficiently hydrolysing esters, carbamates and phosphotriesters. In fact, an analogue of diuron, in which the C-N bond was replaced by a C-O bond, was found to be turned over as efficiently as diuron, suggesting that the substrate specificity is predominantly determined by steric factors. The discovery of PuhA and PuhB on separate continents, and the absence of any other close homologues in the available sequence databases, poses a challenging question regarding the evolutionary origins of these enzymes.
Khurana
Jeevan L
JL
CSIRO Entomology, Canberra, ACT 2601, Australia.
Jackson
Colin J
CJ
Scott
Colin
C
Pandey
Gunjan
G
Horne
Irene
I
Russell
Robyn J
RJ
Herlt
Anthony
A
Easton
Christopher J
CJ
Oakeshott
John G
JG
D000581:Amidohydrolases / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D001673:Biodegradation, Environmental; D002384:Catalysis; D003001:Cloning, Molecular; D004237:Diuron / Q000378:metabolism; D019143:Evolution, Molecular; D006540:Herbicides / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology* / Q000235:genetics
D006540:Herbicides; D004237:Diuron; D000581:Amidohydrolases; C433926:phenylurea hydrolase