Structure of PhnP, a phosphodiesterase of the carbon-phosphorus lyase pathway for phosphonate degradation.
Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent hydrolase of the beta-lactamase superfamily. Screening with a wide array of hydrolytically sensitive substrates indicated that PhnP is an enzyme with phosphodiesterase activity, having the greatest specificity toward bis(p-nitrophenyl)phosphate and 2',3'-cyclic nucleotides. No activity was observed toward RNA. The metal ion dependence of PhnP with bis(p-nitrophenyl)phosphate as substrate revealed a distinct preference for Mn(2+) and Ni(2+) for catalysis, whereas Zn(2+) afforded poor activity. The three-dimensional structure of PhnP was solved by x-ray crystallography to 1.4 resolution. The overall fold of PhnP is very similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn(2+) ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn(2+) binding site is also observed, composed of a set of cysteine and histidine residues that are strictly conserved in the PhnP family. This second metal ion site appears to stabilize a structural motif.
From the Departments of Biochemistry, Kingston, Ontario K7L 3N6, Canada.
Kateryna
K
Podzelinska
Chemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Shu-Mei
SM
He
Chemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Matthew
M
Wathier
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Alexander
A
Yakunin
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Michael
M
Proudfoot
Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Bjarne
B
Hove-Jensen
From the Departments of Biochemistry, Kingston, Ontario K7L 3N6, Canada. Electronic address: dlzechel@chem.queensu.ca.
David L
DL
Zechel
From the Departments of Biochemistry, Kingston, Ontario K7L 3N6, Canada. Electronic address: jia@post.queensu.ca.
Zongchao
Z
Jia
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D017931:DNA Primers; D009097:Multienzyme Complexes; D063065:Organophosphonates; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D008190:Lyases; C051328:carbon-phosphorus lyase; D015032:Zinc