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PDBsum entry 3d03
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References listed in PDB file
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Key reference
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Title
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Substrate-Promoted formation of a catalytically competent binuclear center and regulation of reactivity in a glycerophosphodiesterase from enterobacter aerogenes.
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Authors
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K.S.Hadler,
E.A.Tanifum,
S.H.Yip,
N.Mitić,
L.W.Guddat,
C.J.Jackson,
L.R.Gahan,
K.Nguyen,
P.D.Carr,
D.L.Ollis,
A.C.Hengge,
J.A.Larrabee,
G.Schenk.
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Ref.
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J Am Chem Soc, 2008,
130,
14129-14138.
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PubMed id
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Abstract
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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.
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