PhnJ – A novel radical SAM enzyme from the C–P lyase complex

AbstractPhnJ from the C–P lyase complex catalyzes the cleavage of the carbon–phosphorus bond in ribose-1-phosphonate-5-phosphate (PRPn) to produce methane and ribose-1,2-cyclic-phosphate-5-phosphate (PRcP). This protein is a novel radical SAM enzyme that uses glycyl and thiyl radicals as reactive intermediates in the proposed reaction mechanism. The overall reaction is initiated with the reductive cleavage of S-adenosylmethionine (SAM) by a reduced [4Fe–4S]1+-cluster to form an Ado-CH2∙ radical intermediate. This intermediate abstracts the proR hydrogen from Gly-32 of PhnJ to form Ado-CH3 and a glycyl radical. In the next step, there is hydrogen atom transfer from Cys-272 to the Gly-32 radical to generate a thiyl radical. The thiyl radical attacks the phosphorus center of the substrate, PRPn, to form a transient thiophosphonate radical intermediate. This intermediate collapses via homolytic C–P bond cleavage and hydrogen atom transfer from the proS hydrogen of Gly-32 to produce a thiophosphate intermediate, methane, and a radical intermediate at Gly-32. The final product, PRcP, is formed by nucleophilic attack of the C2-hydroxyl on the transient thiophosphate intermediate. This reaction regenerates the free thiol group of Cys-272. After hydrogen atom transfer from Cys-272 to the Gly-32 radical, the entire process is repeated with another substrate molecule without the use of another molecule of SAM or involvement from the [4Fe–4S]-cluster again

Identification of the histidine ligands to the binuclear metal center of phosphotriesterase by site-directed mutagenesis

ABSTRACT: In order to identify which of the seven histidines in phosphotriesterase participate a t the active site/binuclear metal center of the enzyme, site-directed mutagenesis has been employed to change, individually, each of the seven histidine residues to asparagine. In addition, the gene for the wild-type enzyme has been subcloned without its leader sequence behind a modified ribosomal binding site, leading to a 5-fold increase in protein expression. The seven mutants The bacterial phosphotriesterase from Pseudomonas diminuta is a zinc metalloenzyme, which catalyzes the hydrolysis of a variety of organophosphate triesters, including the insecticide paraoxon: The reaction proceeds via an S~2-like mechanism and likely involves an amino acid residue acting as a general base to assist in the deprotonation of an activated water molecule, which then directly attacks the substrate 845-9452. with a variety of different metals. Replacement of the naturally occurring zinc(I1) metal with other divalent cations, such as cadmium(II), cobalt(II), manganese(II), or nickel(II), apparently does not alter the conformation of the protein, since the specific activities of the various metalsubstituted phosphotriesterase derivatives remain the same or are increased significantly over that of the native zinc enzyme High-resolution '13Cd nuclear magnetic resonance (NMR) spectra of the 113Cd-substituted phosphotriesterase show two cadmium resonances at 212 (Ma site) and 116 ppm (MB site) downfield from Cd(C10&, indicating that the chemical environments around the two metal ions are somewhat different from each other Electron paramagnetic resonance spectroscopy of the manganese-substituted phosphotriesterase indicates that the two Mn(I1) ions are present as an antiferromagnetically exchange-coupled binuclear metal complex (Chae et al., 1993). This requires the two metal sites to be in close proximity and 0006-2960/94/0433-4265$04.50/0 I

Mechanism of cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium LT2

ABSTRACT: Cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium (CbiA) is the first glutamine amidotransferase in the anaerobic biosynthetic pathway of vitamin B 12 and catalyzes the ATPdependent synthesis of cobyrinic acid a,c-diamide from cobyrinic acid using either glutamine or ammonia as the nitrogen source. The cbiA gene was cloned, the overexpressed protein was purified to homogeneity, and the kinetic parameters were determined. CbiA is a monomer with K m values of 0.74, 2.7, 53, and 26 200 µM for cobyrinic acid, ATP, glutamine, and ammonia, respectively. Analysis of the glutaminase partial reaction demonstrated that the hydrolysis of glutamine and the synthesis of the cobyrinic acid a,c-diamide product are uncoupled. The time course for the synthesis of the diamide product and positional isotope exchange experiments demonstrate that CbiA catalyzes the sequential amidation of the c-and a-carboxylate groups of cobyrinic acid via the formation of a phosphorylated intermediate. These results support a model for the catalytic mechanism in which CbiA catalyzes the amidation of the c-carboxylate, and then the intermediate is released into solution and binds to the same catalytic site for the amidation of the a-carboxylate. Several conserved residues in the synthetase active site were mutated to address the molecular basis of the amidation order; however, no changes in the order of amidation were obtained. The mutants D45N, D48N, and E90Q have a dramatic effect on the catalytic activity, whereas no effect was found for the mutant D97N. The substitutions by alanine of L47 and Y46 residues specifically decrease the affinity of the enzyme for the c-monoamide intermediate

Transition State Analysis of the Reaction Catalyzed by the Phosphotriesterase from Sphingiobium sp. TCM1

Organophosphorus flame retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the hydrolysis of the unactivated substituents is not known. To interrogate the catalytic hydrolysis mechanism of Sb-PTE, the pH dependence of the reaction and the effects of changing the solvent viscosity were determined. These experiments were complemented by measurement of the primary and secondary 18-oxygen isotope effects on substrate hydrolysis and a determination of the effects of changing the pKa of the leaving group on the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a single group must be ionized for nucleophilic attack and that a separate general acid is not involved in protonation of the leaving group. The Brønsted analysis and the heavy atom kinetic isotope effects are consistent with an early associative transition state with subsequent proton transfers not being rate limiting. A novel binding mode of the substrate to the binuclear metal center and a catalytic mechanism are proposed to explain the unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of organophosphate substrates

A combined experimental-theoretical study of the ligW-catalyzed decarboxylation of 5-carboxyvanillate in the metabolic pathway for lignin degradation

Although it is a member of the amidohydrolase superfamily, LigW catalyzes the nonoxidative decarboxylation of 5-carboxyvanillate to form vanillate in the metabolic pathway for bacterial lignin degradation. We now show that membrane inlet mass spectrometry (MIMS) can be used to measure transient CO₂ concentrations in real time, thereby permitting us to establish that C–C bond cleavage proceeds to give CO₂ rather than HCO₃^– as the initial product in the LigW-catalyzed reaction. Thus, incubation of LigW at pH 7.0 with the substrate 5-carboxyvanillate results in an initial burst of CO₂ formation that gradually decreases to an equilibrium value as CO₂ is nonenzymatically hydrated to HCO₃^–. The burst of CO₂ is completely eliminated with the simultaneous addition of substrate and excess carbonic anhydrase to the enzyme, demonstrating that CO₂ is the initial reaction product. This finding is fully consistent with the results of density functional theory calculations, which also provide support for a mechanism in which protonation of the C5 carbon takes place prior to C–C bond cleavage. The calculated barrier of 16.8 kcal/mol for the rate-limiting step, the formation of the C5-protonated intermediate, compares well with the observed <i>k</i>_cat value of 27 s^–1 for Sphingomonas paucimobilis LigW, which corresponds to an energy barrier of ∼16 kcal/mol. The MIMS-based strategy is superior to alternate methods of establishing whether CO₂ or HCO₃^– is the initial reaction product, such as the use of pH-dependent dyes to monitor very small changes in solution pH. Moreover, the MIMS-based assay is generally applicable to studies of all enzymes that produce and/or consume small-molecule, neutral gases

Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues

ABSTRACT: The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S P -enantiomers over the corresponding R P -enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k cat /K a for the R P -enantiomers of up to 2700-fold but had little effect on k cat /K a for the corresponding S P -enantiomers. The kinetic preferences for the S P -enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k cat /K a for the S P -enantiomers, while the values of k cat /K a for the R P -enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R P -enantiomers were preferred as substrates over the corresponding S P -enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site. The bacterial phosphotriesterase (PTE) 1 catalyzes the cleavage of P-O, P-F, or P-S bonds in a variety of insecticides and organophosphate nerve agents (1, 2). A generalized reaction for the hydrolysis of a simplified organophosphate substrate is illustrated in Scheme 1. Three binding pockets (small, large, and leaVing group) within the active site of PTE, which interact directly with the primary substituents attached to the phosphorus center of substrates and inhibitors, have been identified by X-ray crystallography (3). Previous investigations with chiral and achiral organophosphate triesters have shown that the reaction rates for various substrates with the wild type PTE depend to a large extent on the size and stereochemical arrangement of the substituents attached to the phosphorus core (4, 5). For example, the kinetic constants with the wild type enzyme for the two enantiomers of chiral organophosphate triesters can differ by up to 2 orders of magnitude with a clear preference for the S P -enantiomer over the R P -enantiomer (4). For the generic substrate depicted in Scheme 1, substituent Y would be bound within the large subsite while substituent X would be bound within the small subsite prior to product formation. For chiral compounds, the faster isomer is the one where substituent Y is physically larger than substituent X. An investigation into the origin of the stereoselectivity of PTE by site-directed mutagenesis has demonstrated that the kinetic preference for chiral substrates is dictated to a large extent by the size of the small subsite (6). This subsite enhances the preference for the S P -enantiomer by sterically hindering the binding and/or orientation of the R P -enantiomer to the active site (6). The most significant residues within the small subsite are Gly-60, Ile-106, Phe-132, and Ser-308. Decreasing the size of the small subsite by mutation of the single glycine residue to an alanine residue significantly increased the stereoselectivity for the preferred S P -enantiomer (6). Conversely, enlargement of the small subsite by mutation of Ile-106, Phe-132, or Ser-308 to an alanine decreased the stereoselective preference for the S P -enantiomer (6). However, enlargement of the large subsite, which prefers the bulkier substituent of the substrate during catalysis, by replacing His-254, His-257, Leu-271, or Met-317 with an alanine residue had relatively little effect on the stereose

Protonation of the Binuclear Metal Center within the Active Site of Phosphotriesterase †

ABSTRACT: Phosphotriesterase (PTE) is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphates, including pesticides and chemical warfare agents, at rates approaching the diffusion controlled limit. The catalytic mechanism of this enzyme features a bridging solvent molecule that is proposed to initiate nucleophilic attack at the phosphorus center of the substrate. X-band EPR spectroscopy is utilized to investigate the active site of Mn/Mn-substituted PTE. Simulation of the dominant EPR spectrum from the coupled binuclear center of Mn/Mn-PTE requires slightly rhombic zero-field splitting parameters. Assuming that the signal arises from the S ) 2 manifold, an exchange coupling constant of J ) -2.7 ( 0.2 cm -1 (H ex ) -2JS 1 ‚S 2 ) is calculated. A kinetic pK a of 7.1 ( 0.1 associated with loss in activity at low pH indicates that a protonation event is responsible for inhibition of catalysis. Analysis of changes in the EPR spectrum as a function of pH provides a pK a of 7.3 ( 0.1 that is assigned as the protonation of the hydroxyl bridge. From the comparison of kinetic and spectral pK a values, it is concluded that the loss of catalytic activity at acidic pH results from the protonation of the hydroxide that bridges the binuclear metal center. Phosphotriesterase (PTE) 1 catalyzes the hydrolysis of a wide range of organophosphate esters, including agricultural pesticides and chemical warfare agents (1-3). The enzyme has been isolated from soil bacteria, but the natural substrate for PTE is not known. PTE is a member of the amidohydrolase superfamily, which also includes urease, dihydroorotase, and approximately 30 other enzymes of known specificity (4). The high-resolution X-ray crystal structure of Zn/Zn-PTE reveals that it is a homodimeric protein containing an active site with two divalent metal ions embedded within a ( /R) 8 -barrel motif (5). The R-metal ion is ligated by His-55, His-57, and Asp-301 while the -metal ion is coordinated to His-201 and His-230 as illustrated i

Functional characterization of two PLP-dependent enzymes involved in capsular polysaccharide biosynthesis from campylobacter jejuni

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed

Substrate distortion and the catalytic reaction mechanism of 5-carboxyvanillate decarboxylase

5-Carboxyvanillate decarboxylase (LigW) catalyzes the conversion of 5-carboxyvanillate to vanillate in the biochemical pathway for the degradation of lignin. This enzyme was shown to require Mn2+ for catalytic activity and the kinetic constants for the decarboxylation of 5-carboxyvanillate by the enzymes from Sphingomonas paucimobilis SYK-6 (kcat = 2.2 s–1 and kcat/Km = 4.0 × 104 M–1 s–1) and Novosphingobium aromaticivorans (kcat = 27 s–1 and kcat/Km = 1.1 × 105 M–1 s–1) were determined. The three-dimensional structures of both enzymes were determined in the presence and absence of ligands bound in the active site. The structure of LigW from N. aromaticivorans, bound with the substrate analogue, 5-nitrovanillate (Kd = 5.0 nM), was determined to a resolution of 1.07 Å. The structure of this complex shows a remarkable enzyme-induced distortion of the nitro-substituent out of the plane of the phenyl ring by approximately 23°. A chemical reaction mechanism for the decarboxylation of 5-carboxyvanillate by LigW was proposed on the basis of the high resolution X-ray structures determined in the presence ligands bound in the active site, mutation of active site residues, and the magnitude of the product isotope effect determined in a mixture of H2O and D2O. In the proposed reaction mechanism the enzyme facilitates the transfer of a proton to C5 of the substrate prior to the decarboxylation step

STRENDA DB : enabling the validation and sharing of enzyme kinetics data

Standards for reporting enzymology data (STRENDA) DB is a validation and storage system for enzyme function data that incorporates the STRENDA Guidelines. It provides authors who are preparing a manuscript with a user‐friendly, web‐based service that checks automatically enzymology data sets entered in the submission form that they are complete and valid before they are submitted as part of a publication to a journal