Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase.

A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD(+) and NADP(+), GDH activity was greater in the deaminating reaction with NADP(+) as co-factor and more with NADH in the aminating reaction.

Arginine metabolism in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta.

The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn(2+) and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected.

Crystal structure of methylmalonyl-coenzyme A epimerase from P. shermanii: a novel enzymatic function on an ancient metal binding scaffold.

BACKGROUND: Methylmalonyl-CoA epimerase (MMCE) is an essential enzyme in the breakdown of odd-numbered fatty acids and of the amino acids valine, isoleucine, and methionine. Present in many bacteria and in animals, it catalyzes the conversion of (2R)-methylmalonyl-CoA to (2S)-methylmalonyl-CoA, the substrate for the B12-dependent enzyme, methylmalonyl-CoA mutase. Defects in this pathway can result in severe acidosis and cause damage to the central nervous system in humans. RESULTS: The crystal structure of MMCE from Propionibacterium shermanii has been determined at 2.0 A resolution. The MMCE monomer is folded into two tandem betaalphabetabetabeta modules that pack edge-to-edge to generate an 8-stranded beta sheet. Two monomers then pack back-to-back to create a tightly associated dimer. In each monomer, the beta sheet curves around to create a deep cleft, in the floor of which His12, Gln65, His91, and Glu141 provide a binding site for a divalent metal ion, as shown by the binding of Co2+. Modeling 2-methylmalonate into the active site identifies two glutamate residues as the likely essential bases for the epimerization reaction. CONCLUSIONS: The betaalphabetabetabeta modules of MMCE correspond with those found in several other proteins, including bleomycin resistance protein, glyoxalase I, and a family of extradiol dioxygenases. Differences in connectivity are consistent with the evolution of these very different proteins from a common precursor by mechanisms of gene duplication and domain swapping. The metal binding residues also align precisely, and striking structural similarities between MMCE and glyoxalase I suggest common mechanisms in their respective epimerization and isomerization reactions.

Growth and arginine metabolism of the wine lactic acid bacteria Lactobacillus buchneri and Oenococcus oeni at different pH values and arginine concentrations.

During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth of Lactobacillus buchneri CUC-3 and Oenococcus oeni strains MCW and Lo111 in wine were investigated. In contrast to L. buchneri CUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth of L. buchneri CUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.

Expression, crystallization and preliminary characterization of methylmalonyl coenzyme A epimerase from Propionibacterium shermanii.

Methylmalonyl-CoA epimerase (MMCE) is an enzyme that interconverts the R and S epimers of methylmalonyl-CoA in the pathway that links propionyl-CoA with succinyl-CoA. This is used for both biosynthetic and degradative processes, including the breakdown of odd-numbered fatty acids and some amino acids. The enzyme has been expressed in Escherichia coli both as the native enzyme and as its selenomethionine (SeMet) derivative. Crystals of both forms have been obtained by vapour diffusion using monomethylether PEG 2000 as precipitant. The native MMCE crystals are orthorhombic, with unit-cell parameters a = 56.0, b = 114.0, c = 156.0 A, and the SeMet-MMCE crystals are monoclinic, with unit-cell parameters a = 43.6, b = 78.6, c = 89.4 A, beta = 92.0 degrees; both diffract to better than 2.8 A resolution.

Kinetics of the arginine metabolism of malolactic wine lactic acid bacteria Lactobacillus buchneri CUC-3 and Oenococcus oeni Lo111.

The excretion of citrulline, a precursor of carcinogenic ethyl carbamate, formed from arginine degradation by malolactic bacteria in wine is of toxicological concern. The arginine metabolism of resting cells of Lactobacillus buchneri CUC-3 and Oenococcus oeni Lo1l1 was examined. The citrulline excretion rate was found to be linearly correlated to the arginine degradation rate. It was possible to calculate an arginine to citrulline conversion ratio which could be used to predict the amount of citrulline expected after the degradation of a known quantity of arginine. The conversion ratios determined in this study were similar to data calculated from other authors for fermentations in wine and ranged between 4.0% and 7.7%. Ribose, fructose and glucose inhibited the degradation of arginine in Lact. buchneri CUC-3, and inhibition of arginine degradation by glucose correlated with higher arginine to citrulline conversion ratios. The work presents new results of arginine metabolism in malolactic bacteria and gives starting points for investigations in wine.

Ethyl carbamate precursor citrulline formation from arginine degradation by malolactic wine lactic acid bacteria.

Major commercially available strains for induction of malolactic fermentation in wine were examined for arginine metabolism in a resting cell system at wine pH with the aim of evaluating their ability to excrete and utilize citrulline, a precursor of carcinogenic ethyl carbamate (urethane). All strains tested excreted citrulline from arginine degradation. Citrulline was stored intracellularly during growth in arginine rich medium and was released upon lysis of the cells. All strains were found to degrade citrulline as a sole amino acid and some of them were able to reutilize previously excreted citrulline.

Crystal structures of Bacillus caldovelox arginase in complex with substrate and inhibitors reveal new insights into activation, inhibition and catalysis in the arginase superfamily.

BACKGROUND: Arginase is a manganese-dependent enzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. In ureotelic animals arginase is the final enzyme of the urea cycle, but in many species it has a wider role controlling the use of arginine for other metabolic purposes, including the production of creatine, polyamines, proline and nitric oxide. Arginase activity is regulated by various small molecules, including the product L-ornithine. The aim of these structural studies was to test aspects of the catalytic mechanism and to investigate the structural basis of arginase inhibition. RESULTS: We report here the crystal structures of arginase from Bacillus caldovelox at pH 5.6 and pH 8.5, and of binary complexes of the enzyme with L-arginine, L-ornithine and L-lysine at pH 8.5. The arginase monomer comprises a single compact alpha/beta domain that further associates into a hexameric quaternary structure. The binary complexes reveal a common mode of ligand binding, which places the substrate adjacent to the dimanganese centre. We also observe a conformational change that impacts on the active site and is coupled with the occupancy of an external site by guanidine or arginine. CONCLUSIONS: The structures reported here clarify aspects of the active site and indicate key features of the catalytic mechanism, including substrate coordination to one of the manganese ions and an orientational role for a neighboring histidine residue. Stereospecificity for L-amino acids is found to depend on their precise recognition at the active-site rim. Identification of a second arginine-binding site, remote from the active site, and associated conformational changes lead us to propose a regulatory role for this site in substrate hydrolysis.

The cloning, expression and crystallisation of a thermostable arginase.

The gene for the thermostable arginase from the thermophilic bacterium 'Bacillus caldovelox' has been cloned and sequenced. Expression of recombinant arginase at high levels has been achieved in E. coli using an inducible T7 RNA polymerase-based system. A facile purification procedure incorporating a heat-treatment step yielded 0.2 g of recombinant arginase per litre of induced culture. The kinetic properties of the purified recombinant protein are essentially identical to the native enzyme. The recombinant protein has been crystallised and one crystal form is isomorphous to crystals of the native protein.

Characterisation of arginase from the extreme thermophile 'Bacillus caldovelox'.

A thermostable arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was purified from the extreme thermophile 'Bacillus caldovelox' (DSM 411) by a procedure including DEAE-Sepharose chromatography, and gel filtration, anion exchange and hydrophobic-interaction fast-protein liquid chromatography, with substantial retention of the metal ion cofactor. The purified enzyme is a hexamer with a subunit Mr of 31,000 +/- 2000 and contains greater than or equal to 1 Mn atom per subunit. Maximum activation on incubation with Mn2+ is 29%. Activity is optimal at pH 9 and at 60 degrees C the Km for arginine is 3.4 mM and Ki(ornithine) is 0.55 mM. Incubation in 0.1 M Mops/NaOH buffer (pH 7) causes rapid inactivation at 60 degrees C (t1/2 (half life) = 4.5 min) and individually 0.1 mM Mn2+ or 1 mg/ml BSA (bovine serum albumin) increase the t1/2 of arginase activity 4-fold, but combined they produce greater than 1000-fold increase and a t1/2 = 105 min at 95 degrees C. Aspartic acid and other species that bind Mn2+ can replace BSA, and it is suggested that arginase can be inactivated by free Mn2+. A strong chelating agent causes inactivation without subunit dissociation, but arginase dissociates rapidly at pH 2.5. Reassociation occurs at pH 9 and is unusual in that it does not require Mn2+.

Adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. Active holoenzyme produced from Escherichia coli.

The linked structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been altered by site-directed mutagenesis and placed under the control of an inducible phage-T7-specific plasmid promoter in Escherichia coli. Conditions have been found under which both alpha- and beta-subunits are produced in soluble form, in near 1:1 ratio, and assemble to form apo-mutase totalling about 5% of the total cellular protein. Methylmalonyl-CoA mutase purified from these cells could be readily converted into the holoenzyme by addition of adenosylcobalamin. The active holoenzyme apparently crystallizes in the same space group as an inactive corrinoid-containing form of the enzyme obtained previously.

Stability and substrate specificity of a beta-glucosidase from the thermophilic bacterium Tp8 cloned into Escherichia coli.

A facile isolation of beta-glucosidase (EC 3.2.1.21) from Escherichia coli containing the recombinant plasmid pNZ1001 carrying a beta-glucosidase gene from the extremely thermophilic anaerobic bacterium Tp8 is reported. The enzyme was purified to homogeneity by anion-exchange chromatography and steric exclusion HPLC following thermal denaturation/precipitation of heat-labile E. coli proteins. The enzyme had a broad specificity for beta-D-glucosides, galactosides, fucosides, and xylosides. Action on aryl-beta-D-glycosides of glucose, galactose, and fucose was characterized by low Km and high Kcat/Km values compared with disaccharide substrates for which specificity decreased in the order laminaribiose, sophorose, cellobiose, beta-gentiobiose, lactose. Galactono-1-4-lactone, glucono-1-5-lactone, and 1-O-methyl-beta-D-glucose were competitive inhibitors with Ki values of 1.6, 0.09, and 17.5 mM, respectively. The enzyme was remarkably stable to detergents, urea, and organic solvents. Thermostability was greatest at the pH activity optimum (pH 6.0-6.5) and half-life (t1/2) values were 11 min at 90 degrees C, 105 min at 85 degrees C, and 900 min at 80 degrees C. Activity was destabilized by Sr2+, Co2+, Ca2+, Mg2+, and Mn2+, but t1/2 increased in the presence of substrates or competitive inhibitors. Activation energy, Ea, was 54.3 kJ.mol-1. A free thiol group(s) was required for full activity, this being rapidly lost in the presence of Hg2+ or N-ethyl maleimide.

Determination of agmatine, arginine, citrulline and ornithine by reversed-phase liquid chromatography using automated pre-column derivatization with o-phthalaldehyde.

A method is presented for the pre-column derivatization of agmatine, arginine, citrulline or ornithine with o-phthalaldehyde-2-mercaptoethanol, and subsequent separation of the derivatives by reversed-phase liquid chromatography. Fluorescent response is linear from 10 to 150 pmol of injected analyte and detection limits range from 28 to 100 fmol. Response factors relative to the internal standard, homocysteic acid, were 1.16 (agmatine and arginine), 1.03 (citrulline) and 0.34 (ornithine). The applicability of the method to the measurement of arginase, arginine deaminase, arginine decarboxylase and other enzyme activities in bacterial extracts was examined.

Purification and properties of a stable beta-glucosidase from an extremely thermophilic anaerobic bacterium.

A beta-glucosidase (EC 3.2.1.21) was purified to homogeneity from cell-free extracts of an extremely thermophilic anaerobic bacterium. The enzyme has an Mr of 43,000 as determined by molecular-exclusion chromatography, has a pI of 4.55 and shows optimum activity at pH 6.2. The enzyme is active against a wide range of aryl beta-glycosides and beta-linked disaccharides, with beta-galactosidase activity only slightly less than beta-glucosidase activity, and significant beta-xylosidase activity. Lineweaver-Burk plots for p-nitrophenyl beta-glucoside, o-nitrophenyl beta-glucoside and cellobiose substrates are biphasic concave-downwards. Inhibition of the beta-glucosidase by substrates and glucose is negligible. Thermal inactivation follows first-order kinetics, with t1/2 (65 degrees C) 45 h, t1/2 (75 degrees C) 47 min and t1/2 (85 degrees C) 1.4 min and a deactivation energy of 380 kJ/mol at pH 6.2. At pH 7.0, which is the optimum pH for thermostability, t1/2 (75 degrees C) is 130 min. At 75 degrees C, at pH 6.2, the thermostability is enhanced about 8-fold by 10% (w/v) glycerol, about 6-fold by 0.2 M-cellobiose and about 3-fold by 5 mM-dithiothreitol and 5 mM-2-mercaptoethanol.