title: Structure and mechanism of PhnP, a phosphodiesterase of the carbon-phosphorus lyase pathway.
abstract: PhnP is a phosphodiesterase that plays an important role within the bacterial carbon-phosphorus lyase (CP-lyase) pathway by recycling a "dead-end" intermediate, 5-phospho-α-d-ribosyl 1,2-cyclic phosphate, that is formed during organophosphonate catabolism. As a member of the metallo-β-lactamase superfamily, PhnP is most homologous in sequence and structure to tRNase Z phosphodiesterases. X-ray structural analysis of PhnP complexed with orthovanadate to 1.5 Å resolution revealed this inhibitor bound in a tetrahedral geometry by the two catalytic manganese ions and the putative general acid residue H200. Guided by this structure, we probed the contributions of first- and second-sphere active site residues to catalysis and metal ion binding by site-directed mutagenesis, kinetic analysis, and ICP-MS. Alteration of H200 to alanine resulted in a 6-33-fold decrease in k(cat)/K(M) with substituted methyl phenylphosphate diesters with leaving group pK(a) values ranging from 4 to 8.4. With bis(p-nitrophenyl)phosphate as a substrate, there was a 10-fold decrease in k(cat)/K(M), primarily the result of a large increase in K(M). Moreover, the nickel ion-activated H200A PhnP displayed a bell-shaped pH dependence for k(cat)/K(M) with pK(a) values (pK(a1) = 6.3; pK(a2) = 7.8) that were comparable to those of the wild-type enzyme (pK(a1) = 6.5; pK(a2) = 7.8). Such modest effects are counter to what is expected for a general acid catalyst and suggest an alternate role for H200 in this enzyme. A Brønsted analysis of the PhnP reaction with a series of substituted phenyl methyl phosphate esters yielded a linear correlation, a β(lg) of -1.06 ± 0.1, and a Leffler α value of 0.61, consistent with a synchronous transition state for phosphoryl transfer. On the basis of these data, we propose a mechanism for PhnP.
authors.affiliation: Department of Chemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Shu-Mei
authors.initials: SM
authors.lastname: He
authors.forename: Matthew
authors.initials: M
authors.lastname: Wathier
authors.forename: Kateryna
authors.initials: K
authors.lastname: Podzelinska
authors.forename: Matthew
authors.initials: M
authors.lastname: Wong
authors.forename: Fern R
authors.initials: FR
authors.lastname: McSorley
authors.forename: Alemayehu
authors.initials: A
authors.lastname: Asfaw
authors.forename: Bjarne
authors.initials: B
authors.lastname: Hove-Jensen
authors.forename: Zongchao
authors.initials: Z
authors.lastname: Jia
authors.forename: David L
authors.initials: DL
authors.lastname: Zechel
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D029968:Escherichia coli Proteins; C558857:PhnP protein, E.coli; D010727:Phosphoric Diester Hydrolases