Kinetic and mechanistic characterization of the formyl-CoA transferase from Oxalobacter formigenes

Oxalobacter formigenes is an obligate anaerobe that colonizes the human gastrointestinal tract and employs oxalate breakdown to generate ATP in a novel process involving the interplay of two coupled enzymes and a membrane-bound oxalate:formate antiporter. Formyl-CoA transferase is a critical enzyme in oxalate-dependent ATP synthesis and is the first Class III CoA-transferase for which a high resolution, three-dimensional structure has been determined (Ricagno, S., Jonsson, S., Richards, N., and Lindqvist, Y. (2003) EMBO J. 22, 3210-3219). We now report the first detailed kinetic characterizations of recombinant, wild type formyl-CoA transferase and a number of site-specific mutants, which suggest that catalysis proceeds via a series of anhydride intermediates. Further evidence for this mechanistic proposal is provided by the x-ray crystallographic observation of an acylenzyme intermediate that is formed when formyl-CoA transferase is incubated with oxalyl-CoA. The catalytic mechanism of formyl-CoA transferase is therefore established and is almost certainly employed by all other members of the Class III CoA-transferase family.     

Crystal structures and kinetic studies of human Kappa class glutathione transferase provide insights into the catalytic mechanism

GSTs (glutathione transferases) are a family of enzymes that primarily catalyse nucleophilic addition of the thiol of GSH (reduced glutathione) to a variety of hydrophobic electrophiles in the cellular detoxification of cytotoxic and genotoxic compounds. GSTks (Kappa class GSTs) are a distinct class because of their unique cellular localization, function and structure. In the present paper we report the crystal structures of hGSTk (human GSTk) in apo-form and in complex with GTX (S-hexylglutathione) and steady-state kinetic studies, revealing insights into the catalytic mechanism of hGSTk and other GSTks. Substrate binding induces a conformational change of the active site from an 'open' conformation in the apo-form to a 'closed' conformation in the GTX-bound complex, facilitating formations of the G site (GSH-binding site) and the H site (hydrophobic substrate-binding site). The conserved Ser(16) at the G site functions as the catalytic residue in the deprotonation of the thiol group and the conserved Asp(69), Ser(200), Asp(201) and Arg(202) form a network of interactions with γ-glutamyl carboxylate to stabilize the thiolate anion. The H site is a large hydrophobic pocket with conformational flexibility to allow the binding of different hydrophobic substrates. The kinetic mechanism of hGSTk conforms to a rapid equilibrium random sequential Bi Bi model.     

3',5' Cyclic nucleotide phosphodiesterases class III: members, structure, and catalytic mechanism

3',5' Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that were previously divided by their primary structure into two major classes: PDE class I and II. The 3',5' cyclic AMP phosphodiesterase from Escherichia coli encoded by the cpdA gene does not show any homology to either PDE class I or class II enzymes and, therefore, represents a new, third class of PDEs. Previously, information about essential structural elements, substrate and cofactor binding sites, and the mechanism of catalysis was unknown for this enzyme. The present study shows by computational analysis that the enzyme encoded by the E. coli cpdA gene belongs to a family of phosphodiesterases that closely resembles the catalytic machinery known from purple acid phosphatases and several other dimetallophosphoesterases. They share both the conserved sequence motif, D-(X)(n) GD-(X)(n)-GNH[E/D]-(X)(n)-H-(X)(n)-GHXH, which contains the invariant residues forming the active site of purple acid phosphatases, a binuclear Fe(3+)-Me(2+)-containing center, as well as a beta(alpha)beta(alpha)beta motif as a typical secondary structure signature. Furthermore, the known biochemical properties of the bacterial phosphodiesterase encoded by the cpdA gene, such as the requirement of iron ions and a reductant for maintaining its catalytic activity, support this hypothesis developed by computational analysis. In addition, the availability of atomic coordinates for several purple acid phosphatases and related proteins allowed the generation of a three-dimensional model for class III cyclic nucleotide phosphodiesterases.     

Endo-xyloglucan transferase,a new class of transferase involved in cell wall construction

Cell shape in plants is constrained by cell walls, which are thick yet dynamic structures composed of crystalline cellulose microfibrils and matrix polymers. Xyloglucans are the principal component of the matrix polymers and bind tightly to the surface of cellulose microfibrils and thereby cross-link them to form an interwoven xyloglucan-cellulose network structure. Thus, cleavage and reconnection of the cross-links between xyloglucan molecules are required for the rearrangement of the cell wall architecture, the process essential for both cell wall expansion and the wall deposition occurring during cell growth and differentiation. Endoxyloglucan transferase (EXT) is a newly identified class of transferase that catalyzes molecular grafting between xyloglucan molecules. This enzyme catalyzes both endo-type splitting of a xyloglucan molecule and reconnection of a newly generated reducing terminus of the xyloglucan to the non-reducing terminus of another xyloglucan molecule, thereby mediating molecular grafting between xyloglucan cross-links in plant cell walls. Molecular cloning and sequencing of EXT-cDNAs derived from five different plant species includingA. thaliana andV. angularis has revealed that the amino acid sequence of the mature protein is extensively conserved in the five different plant species, indicating that EXT protein is ubiquitous among higher plants. This structural study has also disclosed the presence of a group of xyloglucan related proteins (XRPs) with transferase activity in higher plants. Current data strongly suggest that these proteins are involved in a wide spectrum of physiological activities including cell wall expansion and deposition in growing cell walls. Recipient of the Botanical Sociaty Award of Young Scientists, 1993.     

Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism

The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium vinosum, respectively, catalyze the polymerization of beta-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. vinosum synthase suggested that it is structurally homologous to bacterial lipases and allowed identification of active site residues important for catalysis [Jia, Y., Kappock, T. J., Frick, T., Sinskey, A. J., and Stubbe, J. (2000) Biochemistry 39, 3927-3936]. Sequence alignments between the Class I and III synthases revealed similar residues in the R. eutropha synthase. Site-directed mutants of these residues were prepared and examined using HBCoA and a terminally saturated trimer of HBCoA (sT-CoA) as probes. These studies reveal that the R. eutropha synthase possesses an essential catalytic dyad (C319-H508) in which the C319 is involved in covalent catalysis. A conserved Asp, D480, was shown not to be required for acylation of C319 by sT-CoA and is proposed to function as a general base catalyst to activate the hydroxyl of HBCoA for ester formation. Studies of the [(3)H]sT-CoA with wild-type and mutant synthases reveal that 0.5 equiv of radiolabel is covalently bound per monomer of synthase, suggesting that a dimeric form of the enzyme is involved in elongation. These studies, in conjunction with search algorithms for secondary structure, suggest that the Class I and III synthases are mechanistically similar and structurally homologous, despite their physical and kinetic differences.     

Reduction of pentaamminenitrocobalt(III) by hexaaquachromium(II) ion. Reinvestigation of the mechanism

Products of the reduction of [CoNO₂(NH₃)₅]²⁺ by Cr²⁺ were separated and identified under the conditions of [Cr²⁺]₀/[Co(IlI)]₀⩽3 and 0.02 M ⩽[H⁺] ⩽ 0.75 M. The product distribution was dependent on both [Cr²⁺]_o and [H⁺]. The following mechanism is proposed: [CoNO₂(NH₃)₅]²⁺ + Cr²⁺→Co²⁺ + [CrONO(H₂O)₅]²⁺ (i) [CrONO(H₂O)₅]²⁺ + H⁺→[Cr(H₂O)₆]³⁺ + HNO₂ (ii) [CrONO(H₂O)₅]²⁺ + Cr²⁺→Cr(IV) + [CrNO(H₂O)₅]²⁺ (iii) Cr(IV) + Cr²⁺→[(H₂O)₄Cr(OH)₂Cr(H₂O)₄]⁴⁺ (iv) HNO₂ + 2Cr²⁺→[Cr(H₂O)₆]³⁺ + [CrNO(H₂O)₅]²⁺ (v)     

Crystallization of GST2, a human class alpha glutathione transferase

Single crystals of human GST2, a class alpha glutathione transferase have been grown in polyethylene glycol 2000 by the hanging-drop vapour diffusion method. The crystals belong to space group C2 and have cell dimensions a = 100.8 A, b = 95.4 A, c = 105.2 A and beta = 92.4 degrees. The X-ray diffraction pattern extends to better than 3 A resolution.     

Oxalate consumption by lactobacilli: evaluation of oxalyl-CoA decarboxylase and formyl-CoA transferase activity in Lactobacillus acidophilus

AIMS: This study was undertaken to evaluate the oxalate-degrading activity in several Lactobacillus species widely used in probiotic dairy and pharmaceutical preparations. Functional characterization of oxalyl-CoA decarboxylase and formyl-CoA transferase in Lactobacillus acidophilus was performed in order to assess the possible contribution of Lactobacillus in regulating the intestinal oxalate homeostasis. METHODS AND RESULTS: In order to determine the oxalate-degrading ability in 60 Lactobacillus strains belonging to 12 species, a screening was carried out by using an enzymatic assay. A high variability in the oxalate-degrading capacity was found in the different species. Strains of Lact. acidophilus and Lactobacillus gasseri showed the highest oxalate-degrading activity. Oxalyl-CoA decarboxylase and formyl-CoA transferase genes from Lact. acidophilus LA14 were cloned and sequenced. The activity of the recombinant enzymes was assessed by capillary electrophoresis. CONCLUSIONS: Strains of Lactobacillus with a high oxalate-degrading activity were identified. The function and significance of Lact. acidophilus LA14 oxalyl-CoA decarboxylase and formyl-CoA transferase in oxalate catabolism were demonstrated. These results suggest the potential use of Lactobacillus strains for the degradation of oxalate in the human gut. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of probiotic strains with oxalate-degrading activity can offer the opportunity to provide this capacity to individuals suffering from an increased body burden of oxalate and oxalate-associated disorders.     

Investigation of the role of conserved residues Ser13, Asn48 and Pro49 in the catalytic mechanism of the tau class glutathione transferase from Glycine max

Plant glutathione transferases (GSTs) play a key role in the metabolism of various xenobiotics. In this report, the catalytic mechanism of the tau class GSTU4-4 isoenzyme from Glycine max (GmGSTU4-4) was investigated by site-directed mutagenesis and steady-state kinetic analysis. The catalytic properties of the wild-type enzyme and three mutants of strictly conserved residues (Ser13Ala, Asn48Ala and Pro49Ala) were studied in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction. The results showed that the mutations significantly affect substrate binding and specificity. The effect of Ser13Ala mutation on the catalytic efficiency of the enzyme could be explained by assuming the direct involvement of Ser13 to the reaction chemistry and the correct positioning of GSH and CDNB in the ternary catalytic complex. Asn48 and Pro49 were found to have a direct role on the structural integrity of the GSH-binding site (G-site). Moreover, mutation of Asn48 and Pro49 residues may bring about secondary effects altering the thermal stability and the catalytic activity (k_cat) of the enzyme without affecting the nature of the rate-limiting step of the catalytic reaction.     

Cysteine residues are not essential for the catalytic activity of human class Mu glutathione transferase M1a-1a

To investigate the possible involvement of a Cys thiol in the catalysis of the human glutathione transferase M1a-1a, we constructed mutants of this enzyme wherein the four Cys residues present in the native enzyme were replaced by Ala residues. Three mutants, one where all four Cys residues had been replaced and two mutants where three out of four Cys residues were changed into Ala, were characterized regarding their catalytic activities with three different substrates as well as by their binding of three different inhibitors. All three Cys-deficient mutant forms of glutathione transferase M1a-1a were catalytically active with the tested substrates and their binding of inhibitors, measured by I50, were not significantly different from the values previously obtained for the wild-type enzyme. We therefore conclude that none of the Cys residues in this class Mu glutathione transferase are directly involved in the catalysis performed by this enzyme.     

Crystallization of a type III chloramphenicol acetyl transferase

A type III variant of chloramphenicol acetyl transferase was purified from Escherichia coli and crystallized in the presence of cobaltic hexamine chloride and 2-methyl-2, 4-pentandiol. Two crystal forms were obtained, one of which proved to be suitable for high-resolution X-ray diffraction studies. The space group of this form is R32 with aR = 74.5 A, alpha R = 92.5 degrees, with a monomer (Mr 25,000) in the asymmetric unit. The crystals diffract to 1.7 A resolution. The crystal symmetry has led to a re-evaluation of the oligomeric symmetry of the enzyme and the proposal that it is a trimer rather than a tetramer, the quaternary structure predicted previously from studies of the association of hybrid s subunits.     

Enzyme-catalyzed mechanism of isoniazid activation in class I and class III peroxidases

There is an urgent need to understand the mechanism of activation of the frontline anti-tuberculosis drug isoniazid by the Mycobacterium tuberculosis catalase-peroxidase. To address this, a combination of NMR spectroscopic, biochemical, and computational methods have been used to obtain a model of the frontline anti-tuberculosis drug isoniazid bound to the active site of the class III peroxidase, horseradish peroxidase C. This information has been used in combination with the new crystal structure of the M. tuberculosis catalase-peroxidase to predict the mode of INH binding across the class I heme peroxidase family. An enzyme-catalyzed mechanism for INH activation is proposed that brings together structural, functional, and spectroscopic data from a variety of sources. Collectively, the information not only provides a molecular basis for understanding INH activation by the M. tuberculosis catalase-peroxidase but also establishes a new conceptual framework for testing hypotheses regarding the enzyme-catalyzed turnover of this compound in a number of heme peroxidases.     

Oxygen exchange between acetate and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans. Implications for the mechanism of CoA-ester hydrolysis.

The exchange of oxygen atoms between acetate, glutaryl-CoA, and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans was analyzed using [(18)O(2)]acetate together with matrix-assisted laser desorption/ionization time of flight mass spectrometry of an appropriate undecapeptide. The exchange reaction was shown to be site-specific, reversible, and required both glutaryl-CoA and [(18)O(2)]acetate. The observed exchange is in agreement with the formation of a mixed anhydride intermediate between the enzyme and acetate. In contrast, with a mutant enzyme, which was converted to a thiol ester hydrolyase by replacement of the catalytic glutamate residue by aspartate, no (18)O uptake from H(2)(18)O into the carboxylate was detectable. This result is in accord with a mechanism in which the carboxylate of aspartate acts as a general base in activating a water molecule for hydrolysis of the thiol ester intermediate. This mechanism is further supported by the finding of a significant hydrolyase activity of the wild-type enzyme using acetyl-CoA as substrate, whereas glutaryl-CoA is not hydrolyzed. The small acetate molecule in the substrate binding pocket may activate a water molecule for hydrolysis of the nearby enzyme-CoA thiol ester.     

An electron-sharing network involved in the catalytic mechanism is functionally conserved in different glutathione transferase classes

In Anopheles dirus glutathione transferase D3-3, there are electrostatic interactions between the negatively charged glutamyl alpha-carboxylate group of glutathione, the positively charged Arg-66, and the negatively charged Asp-100. This ionic interaction is stabilized by a network of hydrogen bonds from Ser-65, Thr-158, Thr-162, and a conserved water-mediated contact. This alternating ionic bridge interaction between negatively and positively charged residues stabilized by a network of hydrogen bonding we have named an electron-sharing network. We show that the electron-sharing network assists the glutamyl alpha-carboxylate of glutathione to function as a catalytic base accepting the proton from the thiol group forming an anionic glutathione, which is a crucial step in the glutathione transferase (GST) catalysis. Kinetic studies demonstrate that the mutation of electron-sharing network residues results in a decreased ability to lower the pKa of the thiol group of glutathione. Although the residues that contribute to the electron-sharing network are not conserved in the primary sequence, structural characterizations indicate that the presence of the network can be mapped to the same region in all GST classes. A structural diversification but functional conservation suggests a significant role for the electron-sharing network in catalysis as the purpose was maintained during the divergent evolution of GSTs. This network appears to be a functionally conserved motif that contributes to the "base-assisted deprotonation" model suggested to be essential for the glutathione ionization step of the catalytic mechanism.     

The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase

Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities.     

Mutational analysis of the catalytic domain of O-linked N-acetylglucosaminyl transferase

O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT (CD I and CD II), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues (two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CD I and CD II, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades.     

Structural basis for substrate binding and the catalytic mechanism of type III pantothenate kinase

Pantothenate kinase (PanK) catalyzes the first step of the universal five-step coenzyme A (CoA) biosynthetic pathway. The recently characterized type III PanK (PanK-III, encoded by the coaX gene) is distinct in sequence, structure and enzymatic properties from both the long-known bacterial type I PanK (PanK-I, exemplified by the Escherichia coli CoaA protein) and the predominantly eukaryotic type II PanK (PanK-II). PanK-III enzymes have an unusually high Km for ATP, are resistant to feedback inhibition by CoA, and are unable to utilize the N-alkylpantothenamide family of pantothenate analogues as alternative substrates, thus making type III PanK ineffective in generating CoA analogues as antimetabolites in vivo. Previously, we reported the crystal structure of the PanK-III from Thermotoga maritima and identified it as a member of the "acetate and sugar kinase/heat shock protein 70/actin" (ASKHA) superfamily. Here we report the crystal structures of the same PanK-III in complex with one of its substrates (pantothenate), its product (phosphopantothenate) as well as a ternary complex structure of PanK-III with pantothenate and ADP. These results are combined with isothermal titration calorimetry experiments to present a detailed structural and thermodynamic characterization of the interactions between PanK-III and its substrates ATP and pantothenate. Comparison of substrate binding and catalytic sites of PanK-III with that of eukaryotic PanK-II revealed drastic differences in the binding modes for both ATP and pantothenate substrates, and suggests that these differences may be exploited in the development of new inhibitors specifically targeting PanK-III.