TY - JOUR
T1 - Characterization of the phenylurea hydrolases A and B
T2 - Founding members of a novel amidohydrolase subgroup
AU - Khurana, Jeevan L.
AU - Jackson, Colin J.
AU - Scott, Colin
AU - Pandey, Gunjan
AU - Horne, Irene
AU - Russell, Robyn J.
AU - Herlt, Anthony
AU - Easton, Christopher J.
AU - Oakeshott, John G.
PY - 2009/3/1
Y1 - 2009/3/1
N2 - 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 Bronsted 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.
AB - 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 Bronsted 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.
KW - Diuron
KW - Enzymatic bioremediation
KW - Herbicide degradation
KW - Phenylurea hydrolase
UR - http://www.scopus.com/inward/record.url?scp=61449205250&partnerID=8YFLogxK
U2 - 10.1042/BJ20081488
DO - 10.1042/BJ20081488
M3 - Article
SN - 0264-6021
VL - 418
SP - 431
EP - 441
JO - Biochemical Journal
JF - Biochemical Journal
IS - 2
ER -