A novel hyperthermophilic archaeal glyoxylate reductase from Thermococcus litoralis. Characterization, gene cloning, nucleotide sequence and expression in Escherichia coli.

A novel NADH-dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH-dependent glyoxylate reductase was purified approximately 560-fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of approximately 37 kDa. The optimum pH and temperature for enzyme activity were approximately 6.5 and 90 degrees C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 degrees C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was approximately one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The Km values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mM, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36,807. The amino-acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dye-affinity chromatography were needed.     

Purification and characterization of hydroxypyruvate reductase from the facultative methylotroph Methylobacterium extorquens AM1.

Hydroxypyruvate reductase was purified to homogeneity from the facultative methylotroph Methylobacterium extorquens AM1. It has a molecular mass of about 71 kDa, and it consists of two identical subunits with a molecular mass of about 37 kDa. This enzyme uses both NADH (Km = 0.04 mM) and NADPH (Km = 0.06 mM) as cofactors, uses hydroxypyruvate (Km = 0.1 mM) and glyoxylate (Km = 1.5 mM) as the only substrates for the forward reaction, and carries out the reverse reaction with glycerate (Km = 2.6 mM) only. It was not possible to detect the conversion of glycolate to glyoxylate, a proposed role for this enzyme. Kinetics and inhibitory studies of the enzyme from M. extorquens AM1 suggest that hydroxypyruvate reductase is not a site for regulation of the serine cycle at the level of enzyme activity.     

Purification and characterization of glycerate kinase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2.

The glycerate kinase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity and characterized, the first time for an enzyme from a methylotroph. The enzyme was a monomer with a molecular mass about 41-52 kDa. The enzyme was stable against heating at 35 degrees C for 30 min at pH values over 6-10. Maximum activity was observed at pH 8.0 and around 50 degrees C. The Km values for D-glycerate and ATP were 0.13 mM and 0.13 mM, respectively. The enzyme showed high specificity for D-glycerate, and was activated by potassium and ammonium ions. The reaction product of the enzyme was identified as 2-phosphoglycerate.     

Purification and characterization of a novel NADPH(NADH)-dependent glyoxylate reductase from spinach leaves. Comparison of immunological properties of leaf glyoxylate reductase and hydroxypyruvate reductase.

A novel reductase displaying high specificity for glyoxylate and NADPH was purified 3343-fold from spinach leaves. The enzyme was found to be an oligomer of about 125 kDa, composed of four equal subunits of 33 kDa each. A Km for glyoxylate was about 14-fold lower with NADPH than with NADH (0.085 and 1.10 mM respectively), but the maximal activity, 210 mumol/min per mg of protein, was similar with either cofactor. Km values for NADPH and NADH were 3 and 150 microM respectively. Optimal rates with either NADPH or NADH were found in the pH range 6.5-7.4. The enzyme also showed some reactivity towards hydroxypyruvate with rates less than 2% of those observed for glyoxylate. Results of immunological studies, using antibodies prepared against either glyoxylate reductase or spinach peroxisomal hydroxypyruvate reductase, suggested substantial differences in molecular structure of the two proteins. The high rates of NADPH(NADH)-glyoxylate reductase in crude leaf extracts of spinach, wheat and soya bean (30-45 mumol/h per mg of chlorophyll) and its strong affinity for glyoxylate suggest that the enzyme may be an important side component of photorespiration in vivo. In leaves of nitrogen-fixing legumes, this reductase may also be involved in ureide breakdown, utilizing the glyoxylate produced during allantoate metabolism.     

Biochemical evidence that Escherichia coli hyi (orf b0508, gip) gene encodes hydroxypyruvate isomerase

We found a significant activity of hydroxypyruvate isomerase in Escherichia coli clone cells harboring an E. coli gene (called orf b0508 or gip), which is located downstream of the glyoxylate carboligase gene. We newly designated the gene hyi. The enzyme was purified from cell extracts of the E. coli clone. The enzyme had a molecular mass of 58 kDa and was composed of two identical subunits. The optimum pH for the isomerization of hydroxypyruvate was 6.8-7.2. The enzyme required no cofactor. It exclusively catalyzed the isomerization between hydroxypyruvate and tartronate semialdehyde. The apparent K(m) value for hydroxypyruvate was 12.5 mM. The amino acid sequence of E. coli hydroxypyruvate isomerase is highly similar to those of glyoxylate-induced proteins, Gip, found widely from prokaryotes to eukaryotes.     

Some properties of serine: glyoxylate aminotransferase from rye seedlings (Secale cereale L.).

Serine: glyoxylate aminotransferase (EC 2.6.1.45) from rye seedlings catalysed transamination between L-serine and glyoxylate according to the Ping Pong Bi Bi mechanism with double substrate inhibition. As judged from the Km values, L-serine, L-alanine, and L-asparagine served as substrates for the enzyme with glyoxylate, whereas L-alanine and L-asparagine underwent transamination with hydroxypyruvate as acceptor. Pyridoxal phosphate (PLP) seems to be rather loosely bound to the enzyme protein. Aminooxyacetate and D-serine were found to be pure competitive inhibitors of the enzyme, with Ki values of 0.12 microM and 1.6 mM, respectively. Among the PLP inhibitors isonicotinic acid hydrazide and hydroxylamine were far less effective than aminooxyacetate (20% and 70% inhibition at 0.1 mM concentration, respectively). Inhibition by the SH group inhibitors at 1 mM concentration did not exceed 50%. L-Serine distinctly diminished the inhibitory effect of this type inhibitors. Preincubation of the enzyme with glyoxylate distinctly diminished transamination. Glyoxylate limited the inhibitory action of formaldehyde probably by competing for the reactive groups present in the active centre.     

NADH:hydroxypyruvate reductase and NADPH:glyoxylate reductase in algae: partial purification and characterization from Chlamydomonas reinhardtii

Hydroxypyruvate and glyoxylate reductase activities were measured in extracts from the unicellular green algae, Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella miniata, and Dunaliella tertiolecta. Only trace levels of these activities were detectable in the blue-green algae, Anabaena variabilis and Synechococcus leopoliensis. A NADH-dependent hydroxypyruvate reductase was purified 130-fold from Chlamydomonas to a specific activity of 18 mumol NADH oxidized X min-1 X mg protein-1. The pH optimum was 5.0 to 7.0 in the presence of phosphate and the Km(hydroxypyruvate) was 0.05 mM. Substrate inhibition by hydroxypyruvate could be partially relieved by phosphate. The molecular weight, estimated by gel filtration, was 96,000. NADH-dependent glyoxylate reductase activity copurified with the hydroxypyruvate reductase. The Km(glyoxylate) was 10 mM, and the pH optimum was 4.5 to 8.5. A specific NADPH:glyoxylate reductase was also partially purified which did not reduce hydroxypyruvate or pyruvate. The NADPH:glyoxylate reductase had a Km(glyoxylate) of 0.1 mM and a pH optimum of 5.0 to 9.5. These reductases were compared with the pyruvate reductase of Chlamydomonas which also catalyzes the reduction of both hydroxypyruvate and glyoxylate.     

Serine:glyoxylate aminotransferase from the seedlings of rye (Secale cereale L.)

Serine:glyoxylate aminotransferase (EC 2.6.1.45) from green parts of 7-day-old rye seedlings was purified 600-fold. Specific activity of the purified enzyme against L-serine and glyoxylate as substrates was 53.2 mumol/mg protein per minute at 30 degrees C. The enzyme activity with L-alanine or L-asparagine and glyoxylate, or with L-asparagine and hydroxypyruvate was 20% that with L-serine and glyoxylate as the amino group acceptor, whereas with L-alanine or glycine and hydroxypyruvate it was 10% of that value. The reaction rate with pyruvate and L-asparagine, glycine or L-serine was very low. The enzyme was stabilized by the presence of sucrose, pyridoxal phosphate and 2-mercaptoethanol. Molecular sieving of the native enzyme on Sephacryl S-300 gel gave Mr values of 91,200 and 85,000, whereas the molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 43,000, indicating the dimeric structure of the enzyme.     

Purification and some properties of mitochondrial glutamate:glyoxylate aminotransferase and mechanism of its involvement in glycolate pathway in Euglena gracilis z

Euglena contains glutamate:glyoxylate aminotransferase (GGT) both in mitochondria and in cytosol. Both isoforms were separated from each other by DEAE-cellulose chromatography. The mitochondrial enzyme had an apparent Km of 1.9 mM for glutamate and the cytosolic enzyme 52.6 mM. Mitochondrial GGT was further purified by ammonium sulfate fractionation, isoelectric focusing, and gel chromatography. It had a molecular weight of 141,000 and an isoelectric point of pH 4.88; the optimum pH was 8.5. Its apparent Km values for glutamate and for glyoxylate were 2.0 and 0.25 mM, respectively. In addition to glutamate, mitochondrial GGT used 5-hydroxytryptophan, tryptophan, and cysteine as amino donors in the transamination to glyoxylate. Alanine did not support the activity. The relative activity of the enzyme for amino acceptors on the transamination from glutamate was 4-hydroxyphenylpyruvate greater than phenylpyruvate greater than glyoxylate greater than hydroxypyruvate. Pyruvate and 2-oxoglutarate were not used in the reaction. Evidence that GGT functions mainly in the irreversible transamination between glutamate and glyoxylate is presented. The functional significance of GGT in the glycolate pathway of Euglena is also discussed.     

Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.     

Purification and characterization of a recombinant β-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96

A beta-galactosidase isoenzyme, beta-Gall, from Bifidobacterium infantis HL96, was expressed in Escherichia coli and purified to homogeneity. The molecular mass of the beta-Gall subunit was estimated to be 115 kDa by SDS-PAGE. The enzyme appeared to be a tetramer, with a molecular weight of about 470 kDa by native PAGE. The optimum temperature and pH for o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose were 60 degrees C, pH 7.5, and 50 degrees C, pH 7.5, respectively. The enzyme was stable over a pH range of 5.0-8.5, and remained active for more than 80 min at pH 7.0, 50 degrees C. The enzyme activity was significantly increased by reducing agents. Maximum activity required the presence of both Na+ and K+, at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, divalent metal cations, and Cr3+, and to a lesser extent by EDTA and urea. The hydrolytic activity using lactose as a substrate was significantly inhibited by galactose. The Km, and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. beta-Gall possesses strong transgalactosylation activity. The production rate of galactooligosaccharides from 20% lactose at 30 and 60 degrees C was 120 mg/ml, and this rate increased to 190 mg/ml when 30% lactose was used.     

Purification and properties of glyoxylate reductase I from baker's yeast.

The purification and properties of NADPH-linked glyoxylate reductase [EC 1. 1. 1. 79] from baker's yeast were studied. Two active fractions (peak I and peak II) were isolated by DEAE-cellulose column chromatography. The peak I fraction was purified to homogeneity by the criteria of disc gel electrophoresis and tentatively designated glyoxylate reductase I. Its molecular weight was calculated to be 31,000 from gel filtration measurements. The enzyme reduced glyoxylate 7 times faster than hydroxypyruvate and was specific for NADPH. The enzyme showed optimum activity between pH 5.5 and 7.2. The Michaelis constants for glyoxylate and NADPH were found to be 13 mM and 4 microM, respectively. The enzymic activity was not significantly affected by anions, except for nitrate and iodide, which were inhibitory.     

Purification and characterization of an extracellular beta-glucosidase produced by Phoma sp. KCTC11825BP isolated from rotten mandarin peel

A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.     

Identification of a novel glyoxylate reductase supports phylogeny-based enzymatic substrate specificity prediction

Phylogenetic analysis of the superfamily of D-2-hydroxyacid dehydrogenases identified the previously unrecognized cluster of glyoxylate/hydroxypyruvate reductases (GHPR). Based on the genome sequence of Rhizobium etli, the nodulating endosymbiont of the common bean plant, we predicted a putative 3-phosphoglycerate dehydrogenase to exhibit GHPR activity instead. The protein was overexpressed and purified. The enzyme is homodimeric under native conditions and is indeed capable of reducing both glyoxylate and hydroxypyruvate. Other substrates are phenylpyruvate and ketobutyrate. The highest activity was observed with glyoxylate and phenylpyruvate, both having approximately the same kcat/Km ratio. This kind of substrate specificity has not been reported previously for a GHPR. The optimal pH for the reduction of phenylpyruvate to phenyllactate is pH 7. These data lend support to the idea of predicting enzymatic substrate specificity based on phylogenetic clustering.     

Structural, kinetic, and renaturation properties of an induced hydroxypyruvate reductase from Pseudomonas acidovorans.

A hydroxypyruvate reductase has been induced in Pseudomonas acidovorans by growth on glyoxylate. The enzyme has been purified to homogeneity as assessed by the criteria of analytical ultracentrifugation and analytical disc gel electrophoresis. It has a molecular weight of approximately 85,000 and is composed of two identical subunits. The subunits are not interconnected by disulfide bonds although the enzyme has 4 mol of half-cystine per mol of enzyme. The enzyme catalyzes the reversible conversion of hydroxypyruvate to D(minus)-glycerate in the presence of NADH. Glyoxylate cannot replace hydroxypyruvate as a substrate and is a competitive inhibitor of hydroxypyruvate reduction. The activity of the enzyme toward hydroxypyruvate is anion-modulated; the activity of the enzyme toward D(minus)-glycerate is unaffected by anions but is increased by tris-(hydroxymethyl)aminomethane. The subunits of the induced hydroxypyruvate reductase can be renatured. After the enzyme is dissociated in solutions of 6.0 M guanidine hydrochloride containing 0.1 M 2-mercaptoethanol, optimum renaturation occurs when subunits are diluted into a renaturation solvent consisting of 0.04 M Trischloride, pH 7.4, containing 25% glycerol, 25 mM 2-mercaptoethanol, and 0.14 MM NADH. NAD is an inhibitor of renaturation and therefore cannot substitute for NADH. The optimal temperature of dilution and subsequent incubation is 15 degrees, and increases in protein concentration up to 1.2 mg/ml, the highest concentration tested, improve both the rate of renaturation and the yield of active material. The half-time of renaturation at a protein concentration of 1.2 mg/ml was 1 min. The kinetics of renaturation is second order, i.e., is compatible with a bimolecular reaction preducted by the association of two similar subunits. The physical and kinetic parameters of the renatured protein are the same as those of the native enzyme.     

Characterization of tyrosinase and accompanying laccase from Amorphophallus campanulatus

Tyrosinase and laccase activities were detected in the corm of Amorphophallus campanulatus after extraction with ethanol followed by ammonium sulphate precipitation (20-60%) and dialysis against 10 mM Na2HPO4 buffer at pH 7.0. Tyrosinase was found to be the predominant enzyme exhibiting mono- and di-phenolase activities, specificity for L-DOPA as substrate, optimum pH being 6.0, optimum temperature at 40 degrees C and Km at 1.05 mM. Laccase showed substrate specificity for p-phenylenediamine (p-PD), Km at 2.7 mM, optimum pH being 5.0 and was inactivated above 40 degrees C. Three isoforms of tyrosinase were detected on SDS-PAGE with apparent molecular mass approximately 127, 31 and 27 kDa respectively. On staining sections of A. campanulatus with L-DOPA as substrate and 3-methyl benzothiazolinone hydrazone (MBTH) for colour development, tyrosinase was detected in the intercellular spaces of the plant tissue. The cytosolic region did not show any colour indicating the absence of the enzyme.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Purification and characterization of myrosinase from horseradish (Armoracia rusticana) roots.

Myrosinase (beta-thioglucoside glucohydrolase; EC 3.2.3.147) from horseradish (Armoracia rusticana) roots was purified to homogeneity by ammonium sulfate fractionation, Q-sepharose, and concanavalin A sepharose affinity chromatography. The purified protein migrated as a single band with a mass of about 65 kDa on SDS-polyacrylamide gel electrophoresis. Using LC-MS/MS, this band was identified as myrosinase. Western blot analysis, using the anti-myrosinase monoclonal antibody 3D7, showed a single band of about 65 kDa for horseradish crude extract and for the purified myrosinase. The native molecular mass of the purified myrosinase was estimated, using gel filtration, to be about 130 kDa. Based on these data, it appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa. The enzyme exhibited high activity at broad pH (pH 5.0-8.0) and temperature (37 and 45 degrees C). The purified enzyme remained stable at 4 degrees C for more than 1 year. Using sinigrin as a substrate, the Km and Vmax values for the purified enzyme were estimated to be 0.128 mM and 0.624 micromol min(-1), respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid and was able to breakdown intact glucosinolates in a crude extract of broccoli.     

Purification and properties of two glyoxylate reductases from a species of Pseudomonas

1. Two enzymes that catalyse the reduction of glyoxylate to glycollate have been separated and purified from a species of Pseudomonas. Their molecular weights were estimated as 180000. 2. Reduced nicotinamide nucleotides act as the hydrogen donators for the enzymes. The NADH-linked enzyme is entirely specific for its coenzyme but the NADPH-linked reductase shows some affinity towards NADH. 3. Both enzymes convert hydroxypyruvate into glycerate. 4. The glyoxylate reductases show maximal activity at pH6·0–6·8, are inhibited by keto acids and are strongly dependent on free thiol groups for activity. 5. The Michaelis constants for glyoxylate and hydroxypyruvate were found to be of a high order. 6. The reversibility of the reaction has been demonstrated for both glyoxylate reductases and the equilibrium constants were determined. 7. The reduction of glyoxylate and hydroxypyruvate is not stimulated by anions.     

Affinity interaction of hydroxypyruvate reductase from Methylophilus spp. with Cibacron blue F3GA-derived poly(HEMA EGDMA) microspheres: partial purification and characterization

A methylotrophic hydroxypyruvate reductase was partially purified and characterized from Methylophilus spp. using the biomimetic dye, Cibacron Blue F3FA attached to poly(HEMA-EGDMA) microspheres. The absorption capacities of the dye-affinity microspheres were determined by changing pH and the concentration of the proteins in the adsorption medium. Hydroxypyruvate reductase was desorbed from the dye-affinity support specifically with 2 mM NADH solution. The enzyme was purified 10·4-fold with 47% yield. The molecular mass and subunit molecular mass of the enzyme was estimated to be 75 kDa and 37 kDa on the basis of its mobility in polyacrylamide and SDS-polyacrylamide gels, respectively. This suggested a homogeneous dimer structure. The optimal pH was between 5·0 and 7·0, and the maximum enzyme activity was obtained at 50°C. The K_m values of hydroxpyruvate reductase were 0·222 mM for hydroxpyruvate and 0·067 mM for NADH.