Kieran S. Hadler

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 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.

The organophosphate-degrading enzyme from Agrobacterium radiobacter displays mechanistic flexibility for catalysis.

The OP (organophosphate)-degrading enzyme from Agrobacterium radiobacter (OpdA) is a binuclear metallohydrolase able to degrade highly toxic OP pesticides and nerve agents into less or non-toxic compounds. In the present study, the effect of metal ion substitutions and site-directed mutations on the catalytic properties of OpdA are investigated. The study shows the importance of both the metal ion composition and a hydrogen-bond network that connects the metal ion centre with the substrate-binding pocket using residues Arg254 and Tyr257 in the mechanism and substrate specificity of this enzyme. For the Co(II) derivative of OpdA two protonation equilibria (pKa1 ~5; pKa2 ~10) have been identified as relevant for catalysis, and a terminal hydroxide acts as the likely hydrolysis-initiating nucleophile. In contrast, the Zn(II) and Cd(II) derivatives only have one relevant protonation equilibrium (pKa ~4-5), and the μOH is the proposed nucleophile. The observed mechanistic flexibility may reconcile contrasting reaction models that have been published previously and may be beneficial for the rapid adaptation of OP-degrading enzymes to changing environmental pressures.

The divalent metal ion in the active site of uteroferrin modulates substrate binding and catalysis

The purple acid phosphatases (PAP) are binuclear metallohydrolases that catalyze the hydrolysis of a broad range of phosphomonoester substrates. The mode of substrate binding during catalysis and the identity of the nucleophile is subject to debate. Here, we used native Fe(3+)-Fe(2+) pig PAP (uteroferrin; Uf) and its Fe(3+)-Mn(2+) derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe(2+) by Mn(2+) lowers the reactivity of Uf. However, using stopped-flow measurements it could be shown that this replacement facilitates approximately a ten-fold faster reaction between both substrate and inorganic phosphate with the chromophoric Fe(3+) site. These data also indicate that in both metal forms of Uf, phenyl phosphate hydrolysis occurs faster than formation of a mu-1,3 phosphate complex. The slower rate of interaction between substrate and the Fe(3+) site relative to catalysis suggests that the substrate is hydrolyzed while coordinated only to the divalent metal ion. The likely nucleophile is a water molecule in the second coordination sphere, activated by a hydroxide terminally coordinated to Fe(3+). The faster rates of interaction with the Fe(3+) site in the Fe(3+)-Mn(2+) derivative than the native Fe(3+)-Fe(2+) form are likely mediated via a hydrogen bond network connecting the first and second coordination spheres, and illustrate how the selection of metal ions may be important in fine-tuning the function of this enzyme.

Structural flexibility enhances the reactivity of the bioremediator glycerophosphodiesterase by fine-tuning its mechanism of hydrolysis.

The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) belongs to the family of binuclear metallohydrolases and has attracted recent attention due to its potential in bioremediation. Formation of a catalytically competent binuclear center is promoted by the substrate (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Using the paramagnetic properties of Mn(II), we estimated the K(d) values for the metal ions in the alpha and beta sites to be 29 and 344 microM, respectively, in the absence of a substrate analogue. In its presence, the affinity of the beta site increases substantially (K(d) = 56 microM), while that of the alpha site is not greatly affected (K(d) = 17 microM). Stopped-flow fluorescence measurements identified three distinct phases in the catalytic turnover, associated with the initial binding of substrate to the active site (k(obs1)), the assembly of a catalytically active binuclear center (k(obs2)), and subsequent slower structural rearrangements to optimize catalysis (k(obs3)). These three phases depend on the concentration of substrate ([S]), with k(obs1) and k(obs2) reaching maximum values at high [S] (354 and 38 s(-1), respectively), whereas k(obs3) is reduced as [S] is increased. The k(cat) for the hydrolysis of the substrate bis(para-nitrophenyl) phosphate (approximately 1 s(-1)) gradually increases from the moment of initiating the reaction, reaching a maximum when the structural change associated with k(obs3) is complete. This structural change is mediated via an extensive hydrogen-bond network that connects the coordination sphere with the substrate binding pocket, as demonstrated by mutation of two residues in this network (His81 and His217). The identities of both the substrate and the metal ion also affect interactions within this H-bond network, thus leading to some mechanistic variations. Overall, the mechanism employed by GpdQ is a paradigm of a substrate- and metal-ion-induced fit to optimize catalysis.

The reaction mechanism of the Ga(III)Zn(II) derivative of uteroferrin and corresponding biomimetics

Purple acid phosphatase from pig uterine fluid (uteroferrin), a representative of the diverse family of binuclear metallohydrolases, requires a heterovalent Fe(III)Fe(II) center for catalytic activity. The active-site structure and reaction mechanism of this enzyme were probed with a combination of methods including metal ion replacement and biomimetic studies. Specifically, the asymmetric ligand 2-bis{[(2-pyridylmethyl)-aminomethyl]-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl}-4-methylphenol and two symmetric analogues that contain the softer and harder sites of the asymmetric unit were employed to assess the site selectivity of the trivalent and divalent metal ions using 71Ga NMR, mass spectrometry and X-ray crystallography. An exclusive preference of the harder site of the asymmetric ligand for the trivalent metal ion was observed. Comparison of the reactivities of the biomimetics with Ga(III)Zn(II) and Fe(III)Zn(II) centers indicates a higher turnover for the former, suggesting that the M(III)-bound hydroxide acts as the reaction-initiating nucleophile. Catalytically active Ga(III)Zn(II) and Fe(III)Zn(II) derivatives were also generated in the active site of uteroferrin. As in the case of the biomimetics, the Ga(III) derivative has increased reactivity, and a comparison of the pH dependence of the catalytic parameters of native uteroferrin and its metal ion derivatives supports a flexible mechanistic strategy whereby both the μ-(hydr)oxide and the terminal M(III)-bound hydroxide can act as nucleophiles, depending on the metal ion composition, the geometry of the second coordination sphere and the substrate.

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 mu-hydroxide bridge and reduced reactivity. This is the first study to investigate the electronic structure of an enzyme with a mu-1,1-carboxylate bridged dicobalt(II) center.

Promiscuity comes at a price: Catalytic versatility vs efficiency in different metal ion derivatives of the potential bioremediator GpdQ☆

The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) is a highly promiscuous dinuclear metallohydrolase with respect to both substrate specificity and metal ion composition. While this promiscuity may adversely affect the enzymes catalytic efficiency its ability to hydrolyse some organophosphates (OPs) and by-products of OP degradation have turned GpdQ into a promising candidate for bioremedial applications. Here, we investigated both metal ion binding and the effect of the metal ion composition on catalysis. The prevalent in vivo metal ion composition for GpdQ is proposed to be of the type Fe(II)Zn(II), a reflection of natural abundance rather than catalytic optimisation. The Fe(II) appears to have lower binding affinity than other divalent metal ions, and the catalytic efficiency of this mixed metal center is considerably smaller than that of Mn(II), Co(II) or Cd(II)-containing derivatives of GpdQ. Interestingly, metal ion replacements do not only affect catalytic efficiency but also the optimal pH range for the reaction, suggesting that different metal ion combinations may employ different mechanistic strategies. These metal ion-triggered modulations are likely to be mediated via an extensive hydrogen bond network that links the two metal ion binding sites via residues in the substrate binding pocket. The observed functional diversity may be the cause for the modest catalytic efficiency of wild-type GpdQ but may also be essential to enable the enzyme to evolve rapidly to alter substrate specificity and enhance k(cat) values, as has recently been demonstrated in a directed evolution experiment. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.

Catalytic Mechanisms of Metallohydrolases Containing Two Metal Ions

At least one-third of enzymes contain metal ions as cofactors necessary for a diverse range of catalytic activities. In the case of polymetallic enzymes (i.e., two or more metal ions involved in catalysis), the presence of two (or more) closely spaced metal ions gives an additional advantage in terms of (i) charge delocalisation, (ii) smaller activation barriers, (iii) the ability to bind larger substrates, (iv) enhanced electrostatic activation of substrates, and (v) decreased transition-state energies. Among this group of proteins, enzymes that catalyze the hydrolysis of ester and amide bonds form a very prominent family, the metallohydrolases. These enzymes are involved in a multitude of biological functions, and an increasing number of them gain attention for translational research in medicine and biotechnology. Their functional versatility and catalytic proficiency are largely due to the presence of metal ions in their active sites. In this chapter, we thus discuss and compare the reaction mechanisms of several closely related enzymes with a view to highlighting the functional diversity bestowed upon them by their metal ion cofactors.

The bioremediator glycerophosphodiesterase employs a non-processive mechanism for hydrolysis.

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase that catalyzes the breakdown of a broad range of phosphate ester substrates, and it is of interest for its potential application in the destruction of organophosphate nerve agents and pesticides. The reaction mechanism of GpdQ has been proposed to involve a nucleophilic attack by a terminally bound hydroxide molecule. The hydroxide species bridging the two metal ions is suggested to activate the nucleophile, thus favoring a sequential rather than a processive mechanism of action. Here, the hydrolysis of the two ester bonds in the substrate bis(para-nitrophenyl) phosphate (bpNPP) is probed using (31)P NMR. The kinetic rates measured compare well with those determined spectrophotometrically. Furthermore, the data indicate that the diester bonds are cleaved in two separate (non-processive) reactions, indicating that only a single nucleophile (the terminal hydroxide molecule) is likely to be employed as a nucleophile for GpdQ.

Malonate-bound structure of the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) and characterization of the native Fe2+ metal-ion preference.

The structure of a malonate-bound form of the glycerophosphodiesterase from Enterobacter aerogenes, GpdQ, has been refined at a resolution of 2.2 A to a final R factor of 17.1%. The structure was originally solved to 2.9 A resolution using SAD phases from Zn2+ metal ions introduced into the active site of the apoenzyme [Jackson et al. (2007), J. Mol. Biol. 367, 1047-1062]. However, the 2.9 A resolution was insufficient to discern significant details of the architecture of the binuclear metal centre that constitutes the active site. Furthermore, kinetic analysis revealed that the enzyme lost a significant amount of activity in the presence of Zn2+, suggesting that it is unlikely to be a catalytically relevant metal ion. In this communication, a higher resolution structure of GpdQ is presented in which malonate is visibly coordinated in the active site and analysis of the native metal-ion preference is presented using atomic absorption spectroscopy and anomalous scattering. Catalytic implications of the structure and its Fe2+ metal-ion preference are discussed.