Cysteine synthase of an extremely thermophilic bacterium,Thermus thermophilus HB8

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70 degrees C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80 degrees C with 10 mm dithiothreitol. The enzyme was also quite stable at pH 8-12 (50 degrees C, 30 min). It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435 micromol/min/mg of protein. The apparent Km for sulfide was approximately 50 microM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor. The apo-enzyme showed an apparent Km of 29 microM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.     

Partial purification and some properties of homoserine O-acetyltransferase of a methionine auxotroph of Saccharomyces cerevisiae.

A wild-type strain and six methionine auxotrophs of Saccharomyces cerevisiae were cultured in a synthetic medium supplemented with 0.1 mM L-cysteine or L-methionine and analyzed for the synthesis of homoserine O-acetyltransferase (EC 2.3.1.31). Among them, four mutant strains exhibited enzyme activity in cell extracts. Methionine added to the synthetic medium at concentrations higher than 0.1 mM repressed enzyme synthesis in two of these strains. The enzyme was partially purified (3,500-fold) from an extract of a mutant strain through ammonium sulfate fractionation and chromatography on columns of DEAE-cellulose, Phenyl-Sepharose C1-4B, and Sephadex G-150. The enzyme exhibited optimal pH at 7.5 for activity and at 7.8 for stability. The reaction product was ascertained to be O-acetyl-L-homoserine by confirming that it produced L-homocysteine in an O-acetyl-L-homoserine sulfhydrylase reaction. The Km for L-homoserine was 1.0 mM, and for acetyl coenzyme A it was 0.027 mM. The molecular weight of the enzyme was estimated to be approximately 104,000 by Sephadex G-150 column chromatography and 101,000 by sucrose density gradient centrifugation. The isoelectric point was at pH 4.0. Of the hydroxy amino acids examined, the enzyme showed reactivity only to L-homoserine. Succinyl coenzyme A was not an acyl donor. In the absence of L-homoserine, acetyl coenzyme A was deacylated by the enzyme, with a Km of 0.012 mM. S-Adenosylmethionine and S-adenosylhomocysteine slightly inhibited the enzyme, but methionine had no effect.     

Occurrence of transsulfuration in synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus HB8

A cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine gamma-synthesis with O-acetyl-L-homoserine and L-cysteine as substrates but not beta-synthesis with DL-homocysteine and L-serine (or O-acetyl-L-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cystathionine beta-lyase (EC 4.4.1.8) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) were markedly repressed by L-methionine supplemented to the medium. L-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of O-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase. Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of O-acetylserine sulfhydrylase 2.1- to 2.5-fold. A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of O-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine gamma-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine gamma-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of O-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibited O-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis of O-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine gamma-synthase. Although it has been reported that homocysteine is directly synthesized in T. thermophilus HB27 by the catalysis of O-acetylhomoserine sulfhydrylase on the basis of genetic studies (T. Kosuge, D. Gao, and T. Hoshino, J. Biosci. Bioeng. 90:271-279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in T. thermophilus HB8.     

Comparative characterization of the oah2 gene homologous to the oah1 of Thermus thermophilus HB8

The oah2 gene homologous to the oah1 of Thermus thermophilus HB8 was cloned and sequenced. It comprised 1,236 bp encoding a protein of 412 amino acid residues and was overexpressed. The gene product, also having O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity, was purified to homogeneity and characterized comparatively with the oah1 product. The two proteins shared many characteristics.     

Characterization of O-acetyl-L-serine sulfhydrylase purified from an alkaliphilic bacterium

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) activity was shown to be very high compared with O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity and L-cystathionine cleaving activities, in an extract of cells of an alkaliphilic bacterium grown in a synthetic medium. The synthesis of the first enzyme was repressed by approximately 55% by both L-cystine and L-djenkolic acid added to the medium at a concentration of 0.5 mM, but L-methionine (1 mM) and S-adenosyl-L-methionine (0.5 mM) affected it to lesser extents. Its enzyme activity was inhibited by 25% and 12% by methionine (10 mM) and S-adenosylmethionine (5 mM), respectively. The enzyme was purified from the extract through ammonium sulfate fractionation, heat treatment, and chromatography on columns of DEAE-cellulose, Sephacryl S-300, and Octyl Sepharose CL-4B with a recovery of 21%. Polyacrylamide gel electrophoresis with sodium dodecylsulfate of the preparation obtained finally showed its homogeneity and the molecular mass of 37,000 Da for dissociated subunits. Gel filtration of the enzyme on a Sephacryl S-300 column showed an approximate molecular mass of 72,000 Da, suggesting that the enzyme was comprised of two identical subunits. The enzyme catalyzed the beta-replacement reaction with O-acetylserine as a substrate, and showed no reactivity to other O-substituted amino acids tested. The reaction proceeded best at 40 degrees C (when tested at pH 7.5), and at pH 6.5 (at 40 degrees C). The enzyme kept 90% its activity after incubation at 65 degrees C (at pH 7.5) for 30 min, and more than 90% after 30 min incubation at pHs 7-12 at 30 degrees C. The enzyme had a Km of 4 mM for O-acetyl-L-serine and a Vmax of 37.0 micromol/min/mg of protein, a very low value compared with those of other organisms. However, the content of the enzyme in the extract was calculated to be approximately 3.5% total protein. Sensitivity of the enzyme to carbonyl reagents was very low, although it was shown to have pyridoxal 5'-phosphate as a cofactor by examination of its absorption spectrum. Sulfhydryl reagents tested showed no inhibition. The novelty of this enzyme among analogous sulfhydrylases purified from other organisms was discussed.     

O-Acetylhomoserine sulfhydrylase of the fission yeast Schizosaccharomyces pombe: partial purification, characterization, and its probable role in homocysteine biosynthesis

A crude extract of Schizosaccharomyces pombe cells catalyzed sulfhydrylation of both O-acetyl-L-serine and O-acetyl-L-homoserine with H2S, but did not synthesize cystathionine from O-acetyl-L-homoserine and L-cysteine. The O-acetylhomoserine sulfhydrylase [EC 4.2.99.10] was very unstable; however, it could be stabilized by the addition of 25% (w/w) sucrose or glycerol. The optimal pH for activity was 8.0 and that for stability was 7.0. The enzyme was purified approximately 300-fold from an ammonium sulfate-precipitated fraction. L-Methionine was the most effective inhibitor among the amino acids examined. It inhibited the enzyme competitively with respect to OAH with a Ki value of 2.6 mM. Sulfhydrylase activity was inhibited to various extents by some carbonyl reagents, but sulfhydryl reagents such as p-chloromercuribenzoic acid, 5,5'-dithio-bis(2-nitrobenzoic acid), and monoiodoacetic acid had no inhibitory effect. The enzyme also reacted with O-succinylhomoserine and L-homoserine to synthesize homocysteine directly, but could not utilize cysteine as a co-substrate in place of H2S. In the sulfhydrylation reactions, Km values for the substrates ranged from 10.4-12.5 mM. The enzyme was resolved to the apoenzyme by incubation with phenylhydrazine and reactivated by the addition of pyridoxal 5'-phosphate, whose Km value was 0.083 microM. The molecular weight of the enzyme was estimated to be approximately 186,000 by gel filtration and 170,000 by ultracentrifugation in sucrose density gradients. The isolectric point of the protein was pH 4.1. The characteristics of this enzyme are compared with those of physiologically functional sulfhydrylases reported for other organisms, and the possibility of the enzyme functioning as a homocysteine synthase is discussed.     

Purification and properties of phosphoglycerate kinase from Thermus thermophilus strain HB8.

(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10--20 min at 79 degrees C. (2) Catalytic properties such as pH optimum (pH 6--8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0 The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% alpha-helix and 11% pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.     

Molecular cloning of the Lon protease gene from Thermus thermophilus HB8 and characterization of its gene product.

The gene encoding Lon protease was isolated from an extreme thermophile, Thermus thermophilus HB8. Sequence analysis demonstrated that the T. thermophilus Lon protease gene (TT-lon) contains a protein-coding sequence consisting of 2385 bp which is approximately 56% homologous to the Escherichia coli counterpart. As expected, the G/C content of TT-lon was 68%, which is significantly higher than that of the E. coli lon gene (52% G/C). The amino acid sequence of T. thermophilus Lon protease (TT-Lon) predicted from the nucleotide sequence contained several unique sequences conserved in other Lon proteases: (a) a cysteine residue at the position just before the putative ATP-binding domain; (b) motif A and B sequences required for composition of the ATP-binding domain; and (c) a serine residue at the proteolytic active site. Expression of TT-lon under the control of the T7 promoter in E. coli produced an 89-kDa protein with a yield of approximately 5 mg.L-1. Recombinant TT-Lon (rTT-Lon) was purified to homogeneity by sequential column chromatography. The peptidase activity of rTT-Lon was activated by ATP and alpha-casein. rTT-Lon cleaved succinyl-phenylalanyl-leucyl-phenylalanyl-methoxynaphthylamide much more efficiently than succinyl-alanyl-alanyl-phenylalanyl-methoxynaphthylamide, whereas both peptides were cleaved with comparable efficiencies by E. coli Lon. These results suggest that there is a difference between TT-Lon and E. coli Lon in substrate specificity. rTT-Lon most effectively cleaved substrate peptides at 70 degrees C, which was significantly higher than the optimal temperature (37 degrees C) for E. coli Lon. Together, these results indicate that the TT-lon gene isolated from T. thermophilus HB8 actually encodes an ATP-dependent thermostable protease Lon.     

Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.

2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively.     

Optimization of culture conditions and properties of immobilized sulfide oxidase from Arthrobacter species

Arthrobacter species strain FR-3, isolated from sediments of a swamp, produced a novel serine-type sulfide oxidase. The production of sulfide oxidase was maximal at pH 7.5 and 30 degrees C. Among various carbon and nitrogen sources tested, glucose and yeast extract were found to be the most effective substrates for the secretion of sulfide oxidase. The sulfide oxidase was purified to homogeneity and the molecular weight of the purified enzyme was 43 kDa when estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified sulfide oxidase can be effectively immobilized in DEAE (diethylaminoethyl)-cellulose matrix with a yield of 66%. The purified free and immobilized enzyme had optimum activity at pH 7.5 and 6.0, respectively. Immobilization increases the stability of the enzyme with respect to temperature. The half-life of the immobilized enzyme was 30 min at 45 degrees C, longer than that of the free enzyme (10 min). The purified free sulfide oxidase activity was completely inhibited by 1 mM Co2+ and Zn2+ and sulfhydryl group reagents (para-chloromercuribenzoic acid and iodoacetic acid). Catalytic activity was not affected by 1 mM Ca2+, Mg2+, Na+ and metal-chelating agent (EDTA).     

Purification and Properties of Dextransucrase from Streptococcus mutans

The dextransucrase (EC 2.4.1.5) activity from cell-free culture supernatants of Streptococcus mutans strain 6715 has been purified approximately 1,500-fold by ammonium sulfate precipitation, hydroxylapatite chromatography, and isoelectric focusing. The enzyme was eluted as a single peak of activity from hydroxylapatite, and isoelectric focusing of the resulting preparation gave a single band of dextransucrase activity which focused at a pH of 4.0. The final enzyme preparation contained two distinct, enzymatically active proteins as judged by assay in situ after polyacrylamide gel electrophoresis. One of the proteins represented 90% of the total dextransucrase activity and 53% of the total protein. The molecular weight of the enzyme was estimated by gel filtration to be 94,000. The temperature optimum of the enzyme was broad (34 to 42 C) and its pH range was rather narrow, with optimal activity at pH 5.5. The K(m) for sucrose was 3 mM, and fructose competitively inhibited the enzyme reaction with a K(i) of 27 mM.     

Dirofilaria immitis: comparison of cytosolic and mitochondrial glutamate dehydrogenases

Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8-8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and less than 100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 microM NADH, the Km for the substrate, alpha-ketoglutarate, was equivalent for the two enzymes, but the Km for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 microM NADH, 1 mM ADP approximately doubled and 1 mM ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 mM glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate Km of M-GDH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: effect of N-terminus in thermal activity and stability

Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(?)< 5 min) than the wild-type enzyme (t(?)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(?)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability.     

The bacterium Thermus thermophilus,like hyperthermophilic archaea,uses a two-step pathway for the synthesis of mannosylglycerate

The biosynthetic pathway for the synthesis of the compatible solute alpha-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of alpha-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and D-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90 degrees C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95 degrees C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn(2+) was most effective for MPGS, while Mn(2+), Co(2+), Mg(2+), and to a lesser extent Ni(2+) activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.     

Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh(Tt)) was heterologously overexpressed in Escherichia coli, and the protein (ADH(Tt)) was purified to homogeneity and characterized. ADH(Tt) is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees C, as well as good tolerance to common organic solvents. ADH(Tt) has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, alpha-tetralone, and alpha-methyl and alpha-ethyl benzoylformates to (S)-(-)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-alpha-(trifluoromethyl)benzyl alcohol (93% ee), (S)-alpha-tetralol (>99% ee), methyl (R)-(-)-mandelate (92% ee), and ethyl (R)-(-)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.     

Purification and characterization of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the dromedary camel

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12),a key enzyme ofcarbon metabolism,was purified and characterized to homogeneity from skeletal muscle of Camelusdromedarius.The protein was purified approximately 26.8 folds by conventional ammonium sulphatefractionation followed by Blue Sepharose CL-6B chromatography,and its physical and kinetic propertieswere investigated.The native protein is a homotetramer with an apparent molecular weight of approximately146 kDa.Isoelectric focusing analysis showed the presence of only one GAPDH isoform with an isoelectricpoint of 7.2.The optimum pH of the purified enzyme was 7.8.Studies on the effect of temperature onenzyme activity revealed an optimal value of approximately 28-32 ℃ with activation energy of 4.9 kcal/mol.The apparent K_m values for NAD~ and DL-glyceraldehyde-3-phophate were estimated to be 0.025±0.040mM and 0.21±0.08 mM, respectively. The V_(max) of the purified protein was estimated to be 52.7±5.9 U/mg.These kinetic parameter values were different from those described previously, reflecting protein differencesbetween species.     

Phosphoenolpyruvate carboxylase from the roots of yellow lupin (Lupinus luteus)

A crude preparation of PEP carboxylase (EC 4.1.1.31) from the yellow lupin roots exhibits the pH optimum of activity within the range of 7.4-8.6 and the temperature optimum at 32 - 40 degrees C. Its Km for PEP is 0.1 mM, and Km for HCO3- is 0.7 mM. The affinity of the enzyme towards Mg2+ diminishes with the metal ion concentration. At the concentration of Mg2+ below 0.5 mM Km for Mg2+ is 0.07 mM and at the Mg2+ concentration over 1.5 mM it rises to 0.47 mM. The Hill coefficients are 0.37 and 0.88, respectively. Among several compounds affecting the PEP carboxylase activity, such as organic acids, amino acids, and sugar phosphates, at physiological pH (7.0 and 7.8), malate shows the strongest inhibition of a competitive character, its Ki being 2 mM. Also acidic amino acids strongly inhibit the enzyme activity, aspartate being more effective than glutamate. Glucose 6-phosphate and fructose 1,6-diphosphate markedly activate the enzyme. Both the inhibition by malate, aspartate and glutamate, and the activation by sugar phosphates rises considerably when pH is decreased from 7.8 to 7.0. Malonate scarcely affects the enzyme.     

Purification and properties of D-glyceraldehyde-3-phosphate dehydrogenase from an extreme thermophile, Thermus thermophilus strain HB 8.

1. D-Glyceraldehyde-3-phosphate dehydrogenase from an extreme thermophile, T. thermophilus strain HB8, was purified and crystallized. 2. The enzyme was found to possess remarkable heat stability, being slowly inactivated at 90 degrees C. 3. Basic kinetic constants and pH profile are reported. The enzyme was activated 25-fold by 90 mM NH4Cl, and also by ethanol up to 5-fold at 30 degrees C. 4. The enzyme was found to be far more resistant to urea or sodium dodecylsulfate than the rabbit enzyme. 5. The enzyme was shown to be a tetramer of molecular weight 130000--135000. Amino acid composition analysis revealed no unusual features. Circular dichroic spectra suggested that the contents of the ordered structure of the thermophile enzyme are similar to those of the rabbit enzyme. 6. The other catalytic properties of the thermophile enzyme are discussed in comparison with those of the enzymes from other sources.     

Characterization of a thermoalkalophilic esterase from a novel thermophilic bacterium, Anoxybacillus gonensis G2

Poly-3-hydroxybutyrate (P3HB) degradation capabilities of a novel bacterium, Anoxybacillus gonensis G2, were investigated. Both changes on film surfaces of the solution-cast films monitored by scanning electron microscopy (SEM) and weight loss up to 24% after 72 h exposure to A. gonensis G2 cultures indicated secretion of an active esterase responsible for the degradation of P3HB films. Kinetic parameters, Vmax and Km for the esterase activity of crude enzyme from A. gonensis G2 in the presence of p-nitrophenylbutyrate as substrate were observed as 50 U/L and 0.125 mM, respectively, in 50 mM phosphate buffer, pH 7.5 at 60 degrees C. The stimulation of the activity by Ca2+ is an evidence for the requirement of Ca2+ as a cofactor for the enzyme activity which is a characteristic for lipases/esterases. Inhibition of the esterase activity by metal chelating agents such as ethylenediamine tetraacetate, azide and cyanide has also supported the requirement of a metal ion for the activity. The thermal and pH stability profiles for the enzyme showed that the thermophilic bacterium A. gonensis G2 secretes an extracellular thermoalkalophilic PHB depolymerase active at 60 degrees C, and stable at this temperature for 120 min at pH 7.5 and for 24 h at pH 7.5-9.5 range at 4 degrees C by retaining over 75% of its initial activities.