title: Substrate-promoted formation of a catalytically competent binuclear center and regulation of reactivity in a glycerophosphodiesterase from Enterobacter aerogenes.
abstract: The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous binuclear metallohydrolase that catalyzes the hydrolysis of mono-, di-, and triester substrates, including some organophosphate pesticides and products of the degradation of nerve agents. GpdQ has attracted recent attention as a promising enzymatic bioremediator. Here, we have investigated the catalytic mechanism of this versatile enzyme using a range of techniques. An improved crystal structure (1.9 A resolution) illustrates the presence of (i) an extended hydrogen bond network in the active site, and (ii) two possible nucleophiles, i.e., water/hydroxide ligands, coordinated to one or both metal ions. While it is at present not possible to unambiguously distinguish between these two possibilities, a reaction mechanism is proposed whereby the terminally bound H2O/OH(-) acts as the nucleophile, activated via hydrogen bonding by the bridging water molecule. Furthermore, the presence of substrate promotes the formation of a catalytically competent binuclear center by significantly enhancing the binding affinity of one of the metal ions in the active site. Asn80 appears to display coordination flexibility that may modulate enzyme activity. Kinetic data suggest that the rate-limiting step occurs after hydrolysis, i.e., the release of the phosphate moiety and the concomitant dissociation of one of the metal ions and/or associated conformational changes. Thus, it is proposed that GpdQ employs an intricate regulatory mechanism for catalysis, where coordination flexibility in one of the two metal binding sites is essential for optimal activity.
authors.affiliation: School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: Kieran S
authors.initials: KS
authors.lastname: Hadler
authors.forename: Eric A
authors.initials: EA
authors.lastname: Tanifum
authors.forename: Sylvia Hsu-Chen
authors.initials: SH
authors.lastname: Yip
authors.forename: Natasa
authors.initials: N
authors.lastname: Mitić
authors.forename: Luke W
authors.initials: LW
authors.lastname: Guddat
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Jackson
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Gahan
authors.forename: Kelly
authors.initials: K
authors.lastname: Nguyen
authors.forename: Paul D
authors.initials: PD
authors.lastname: Carr
authors.forename: David L
authors.initials: DL
authors.lastname: Ollis
authors.forename: Alvan C
authors.initials: AC
authors.lastname: Hengge
authors.forename: James A
authors.initials: JA
authors.lastname: Larrabee
authors.forename: Gerhard
authors.initials: G
authors.lastname: Schenk
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D003035:Cobalt / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D021902:Enterobacter aerogenes / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004952:Esters / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008280:Magnetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D017680:Potassium Compounds / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D004952:Esters; D010710:Phosphates; D017680:Potassium Compounds; D003035:Cobalt; C013216:potassium phosphate; D010727:Phosphoric Diester Hydrolases; C038133:glycerophosphodiester phosphodiesterase