James A. Larrabee

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.

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.

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.

Magnetic circular dichroism study of a dicobalt(II) methionine aminopeptidase/fumagillin complex and dicobalt II-II and II-III model complexes.

The dicobalt form of the metallohydrolase methionine aminopeptidase from Escherichia coli (CoCo EcMetAP) has an active site with one 5-coordinate Co (II) and a more weakly bound 6-coordinate Co (II). These metal ions are bridged by two carboxylate amino acid side chains and water or hydroxide, potentially enabling magnetic exchange coupling between the metals. We used variable-temperature, variable-field magnetic circular dichroism to determine whether such coupling occurs. CoCo EcMetAPs MCD spectrum shows distinct d-d transitions at 495 and 567 nm caused by 6- and 5-coordinate Co (II), respectively. The magnetization curves for 5- and 6-coordinate Co (II) are very different, indicating that their electronic ground states vary considerably, ruling out any coupling. When the fungal metabolite fumagillin binds to the CoCoEcMetAP, the qualitative MCD spectrum is unchanged; however, VTVH MCD data show that 5- and 6-coordinate Co (II) ions have similarly shaped magnetization curves, indicating that the Co (II) ions now share the same electronic ground state. Fitting the VTVH MCD data to a model in which dimer wave functions are calculated using a spin Hamiltonian with zero-field splitting showed the Co (II) ions to be weakly ferromagnetically coupled, with J = 2.9 cm (-1). Ferromagnetic coupling is unusual for dinuclear Co (II); therefore, to support the CoCoEcMetAP/fumagillin complex results, we also analyzed VTVH MCD data from a matched pair of dinuclear cobalt complexes, 1 and 2. Complex 1 shares the carboxylate and hydroxide-bridged dicobalt(II) structural motif with the active site of CoCo EcMetAP. Complex 2 contains a nearly isostructural Co (II) ion, but the Co (III) is diamagnetic, so any magnetic coupling is switched off, while the spectral features of the Co (II) ion remain. Magnetization data for 1, fitted to the dimer model, showed that the Co (II) ions were weakly ferromagnetically coupled, with J = 1.7 cm (-1). Magnetization data for Co (II) ions in 2, however, reflect loss of magnetic exchange coupling.

Immobilization of the enzyme GpdQ on magnetite nanoparticles for organophosphate pesticide bioremediation

Annually thousands of people die or suffer from organophosphate (pesticide) poisoning. In order to remove these toxic compounds from the environment, the use of enzymes as bioremediators has been proposed. We report here a Ser127Ala mutant based on the enzyme glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. The mutant, with improved metal binding abilities, has been immobilized using glutaraldehyde on PAMAM dendrimer-modified magnetite nanoparticles. The immobilized system was characterized using elemental analysis as well as infrared, transmission electron and X-ray photoelectron spectroscopies. The amount of GpdQ that was immobilized with the optimized procedure was 1.488 nmol per g MNP. A kinetic assay has been designed to evaluate the activity of the system towards organophosphoester substrates. The specific activity towards BPNPP directly after immobilization was 3.55 μmol mg(-1)min(-1), after one week 3.39 μmol mg(-1)min(-1) and after 120 days 3.36 μmol mg(-1)min(-1), demonstrating that the immobilized enzyme was active for multiple cycles and could be stored on the nanoparticles for a prolonged period.

Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol–Acid Reductoisomerase

Ketol-acid reductoisomerase (KARI) is a Mg(2+) -dependent enzyme in the branched-chain amino acid biosynthesis pathway. It catalyses a complex two-part reaction: an alkyl migration followed by a NADPH-dependent reduction. Both reactions occur within the one active site, but in particular, the mechanism of the isomerisation step is poorly understood. Here, using a combination of kinetic, thermodynamic and spectroscopic techniques, the reaction mechanisms of both Escherichia coli and rice KARI have been investigated. We propose a conserved mechanism of catalysis, whereby a hydroxide, bridging the two Mg(2+) ions in the active site, initiates the reaction by abstracting a proton from the C2 alcohol group of the substrate. While the μ-hydroxide-bridged dimetallic centre is pre-assembled in the bacterial enzyme, in plant KARI substrate binding leads to a reduction of the metal-metal distance with the concomitant formation of a hydroxide bridge. Only Mg(2+) is capable of promoting the isomerisation reaction, likely to be due to non-competent substrate binding in the presence of other metal ions.

CaII binding regulates and dominates the reactivity of a transition-metal-ion-dependent diesterase from Mycobacterium tuberculosis

The diesterase Rv0805 from Mycobacterium tuberculosis is a dinuclear metallohydrolase that plays an important role in signal transduction by controlling the intracellular levels of cyclic nucleotides. As Rv0805 is essential for mycobacterial growth it is a promising new target for the development of chemotherapeutics to treat tuberculosis. The in vivo metal-ion composition of Rv0805 is subject to debate. Here, we demonstrate that the active site accommodates two divalent transition metal ions with binding affinities ranging from approximately 50 nm for Mn(II) to about 600 nm for Zn(II) . In contrast, the enzyme GpdQ from Enterobacter aerogenes, despite having a coordination sphere identical to that of Rv0805, binds only one metal ion in the absence of substrate, thus demonstrating the significance of the outer sphere to modulate metal-ion binding and enzymatic reactivity. Ca(II) also binds tightly to Rv0805 (Kd ≈40 nm), but kinetic, calorimetric, and spectroscopic data indicate that two Ca(II) ions bind at a site different from the dinuclear transition-metal-ion binding site. Ca(II) acts as an activator of the enzymatic activity but is able to promote the hydrolysis of substrates even in the absence of transition-metal ions, thus providing an effective strategy for the regulation of the enzymatic activity.

Use of magnetic circular dichroism to study dinuclear metallohydrolases and the corresponding biomimetics

AbstractMagnetic circular dichroism (MCD) is a convenient technique for providing structural and mechanistic insight into enzymatic systems in solution. The focus of this review is on aspects of geometric and electronic structure that can be determined by MCD, and how this method can further our understanding of enzymatic mechanisms. Dinuclear Co(II) systems that catalyse hydrolytic reactions were selected to illustrate the approach. These systems all contain active sites with similar structures consisting of two Co(II) ions bridged by one or two carboxylates and a water or hydroxide. In most of these active sites one Co(II) is five-coordinate and one is six-coordinate, with differing binding affinities. It is shown how MCD can be used to determine which binding site—five or six-coordinate—has the greater affinity. Importantly, zero-field-splitting data and magnetic exchange coupling constants may be determined from the temperature and field dependence of MCD data. The relevance of these data to the function of the enzymatic systems is discussed.

AIM‐1: An Antibiotic‐Degrading Metallohydrolase That Displays Mechanistic Flexibility

Antibiotic resistance has emerged as a major threat to global health care. This is largely due to the fact that many pathogens have developed strategies to acquire resistance to antibiotics. Metallo-β-lactamases (MBL) have evolved to inactivate most of the commonly used β-lactam antibiotics. AIM-1 is one of only a few MBLs from the B3 subgroup that is encoded on a mobile genetic element in a major human pathogen. Here, its mechanism of action was characterised with a combination of spectroscopic and kinetic techniques and compared to that of other MBLs. Unlike other MBLs it appears that AIM-1 has two avenues available for the turnover of the substrate nitrocefin, distinguished by the identity of the rate-limiting step. This observation may be relevant with respect to inhibitor design for this group of enzymes as it demonstrates that at least some MBLs are very flexible in terms of interactions with substrates and possibly inhibitors.