8 research outputs found
Directed evolution, rational design and mechanistic studies of a phosphohydrolase from Enterobacter aerogenes
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Electronic Structure Analysis of the Dinuclear Metal Center in the Bioremediator Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes
The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous, dinuclear metallohydrolase
that has potential application in the remediation of organophosphate nerve agents and pesticides. GpdQ employs an
unusual reaction mechanism in which the enzyme is predominantly mononuclear in the resting state, and substrate
binding induces the formation of the catalytically competent dinuclear center (Hadler et al. J. Am. Chem. Soc. 2008,
130, 14129). Reactivity is further modulated by the coordination flexibility of Asn80, a ligand that binds to the second,
loosely bound metal ion (Hadler et al. J. Am. Chem. Soc. 2009, 131, 11900). It is proposed that hydrolysis is initiated by
a terminal, metal-bound hydroxide molecule which is activated at unusually low pH by electrostatic/hydrogen bonding
interactions with a bridging hydroxide species. In this study, electronic structure analysis of the dinuclear center is
employed to study the coordination environment of the dinuclear center at the resting and product-bound stage of
catalysis. This is achieved through the use of variable temperature, variable field magnetic circular dichroism
experiments involving the Co(II)-substituted wild type enzyme and its Asn80Asp variant. The data support the above
model for the catalytic mechanism whereby the metal ion-bridging hydroxide molecule activates a terminally bound
hydroxide nucleophile. Replacement of Asn80 by an aspartate residue does prevent coordination flexibility but also
leads to cleavage of the μ-hydroxide bridge and reduced reactivity. This is the first study to investigate the electronic
structure of an enzyme with a μ-1,1-carboxylate bridged dicobalt(II) center
Electronic Structure Analysis of the Dinuclear Metal Center in the Bioremediator Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes
The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous, dinuclear metallohydrolase
that has potential application in the remediation of organophosphate nerve agents and pesticides. GpdQ employs an
unusual reaction mechanism in which the enzyme is predominantly mononuclear in the resting state, and substrate
binding induces the formation of the catalytically competent dinuclear center (Hadler et al. J. Am. Chem. Soc. 2008,
130, 14129). Reactivity is further modulated by the coordination flexibility of Asn80, a ligand that binds to the second,
loosely bound metal ion (Hadler et al. J. Am. Chem. Soc. 2009, 131, 11900). It is proposed that hydrolysis is initiated by
a terminal, metal-bound hydroxide molecule which is activated at unusually low pH by electrostatic/hydrogen bonding
interactions with a bridging hydroxide species. In this study, electronic structure analysis of the dinuclear center is
employed to study the coordination environment of the dinuclear center at the resting and product-bound stage of
catalysis. This is achieved through the use of variable temperature, variable field magnetic circular dichroism
experiments involving the Co(II)-substituted wild type enzyme and its Asn80Asp variant. The data support the above
model for the catalytic mechanism whereby the metal ion-bridging hydroxide molecule activates a terminally bound
hydroxide nucleophile. Replacement of Asn80 by an aspartate residue does prevent coordination flexibility but also
leads to cleavage of the μ-hydroxide bridge and reduced reactivity. This is the first study to investigate the electronic
structure of an enzyme with a μ-1,1-carboxylate bridged dicobalt(II) center
Substrate-Promoted Formation of a Catalytically Competent Binuclear Center and Regulation of Reactivity in a Glycerophosphodiesterase from Enterobacter aerogenes
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 Å 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
Fehlerkultur aus der Sicht von Schülerinnen und Schülern. Der Fragebogen S-UFS: Entwicklung und erste Ergebnisse
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 Å 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
Integrated Genomic Characterization of Papillary Thyroid Carcinoma
Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Here, we describe the genomic landscape of 496 PTCs. We observed a low frequency of somatic alterations (relative to other carcinomas) and extended the set of known PTC driver alterations to include EIF1AX, PPM1D, and CHEK2 and diverse gene fusions. These discoveries reduced the fraction of PTC cases with unknown oncogenic driver from 25% to 3.5%. Combined analyses of genomic variants, gene expression, and methylation demonstrated that different driver groups lead to different pathologies with distinct signaling and differentiation characteristics. Similarly, we identified distinct molecular subgroups of BRAF-mutant tumors, and multidimensional analyses highlighted a potential involvement of onco-miRs in less-differentiated subgroups. Our results propose a reclassification of thyroid cancers into molecular subtypes that better reflect their underlying signaling and differentiation properties, which has the potential to improve their pathological classification and better inform the management of the diseaseclose6