Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803.

NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.     

Formation and dissimilation of oxalacetate and pyruvate Pseudomonas citronellolis grown on noncarbohydrate substrates.

Metabolism of lactate as a carbon source by Pseudomonas citronellolis occurred via a nicotinamide adenine dinucleotide (NAD)-independent L-lactate dehydrogenase, which was present in cells grown on DL-lactate but was not present in cells grown on acetate, aspartate, citrate, glucose, glutamate, or malate. The cells also possessed a constitutive, NAD-independent malate dehydrogenase instead of the conventional NAD-dependent malate dehydrogenase instead of the conventional NAD-dependent enzyme in the tricarboxylic acid cycle. Both enzymes were particulate and used dichlorophenolindo-phenol or oxygen as an electron acceptor. In acetate-grown cells, the activity of pyruvate dehydrogenase and NAD phosphate-linked malate enzyme decreased, cells grown on glucose or lactate. This was consistent with the need to maintain a supply of oxalacetate for metabolism of acetate via the tricarboxylic acid cycle. Changes in enzyme activities suggest that gluconeogenesis from noncarbohydrate carbon sources occurs via the malate enzyme (when oxalacetate decarboxylase is inhibited) or a combination of the NAD-independent malate dehydrogenase and oxalacetate decarboxylase.     

Anaerobic metabolism of the common cockle, Cardium edule. II. Partial purification and properties of lactate dehydrogenase and octopine dehydrogenase. A comparative study.

1. Octopine dehydrogenase and lactate dehydrogenase were purified 190-fold and 10-fold respectively from the adductor muscle of the marine bivalve Cardium edule by gel filtration on Sephadex G-100 and chromatography on DEAE-Sephadex A-50. 2. Lactate dehydrogenase was capable to convert D- and L-lactate, had a molecular weight of about 70 000 and 280 000 daltons, exhibits no distinct pH optimum and was not inhibited by lactate. The enzyme showed apparent Km values of 0.16 mM for pyruvate and 16 mM and 48 mM for D- and L-lactate respectively. 3. In comparison to the purified enzymes from other species, octopine dehydrogenase from Cardium edule showed similar biochemical properties : pH optima of 6.8 and 8.7 respectively, Km values of 0.9 mM (for pyruvate) and 2.0 mM (for arginine), a molecular weight of 37 000 daltons and inhibition by octopine. Electrophoretic studies on standard polyacrylamide gels showed five isoenzymes. 4. The biochemical properties of both dehydrogenases are compared to the conditions in vivo of these animals and the biological role of the octopine dehydrogenase is discussed.     

Electron acquisition system constructed from an NAD-independent D-lactate dehydrogenase and cytochrome c2 in Rhodopseudomonas palustris No. 7

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M(r), about 235,000; subunit M(r) about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50 degrees C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.     

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli.

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, predent in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Stephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.     

Synthesis and localization of L-lactate oxidase in yeasts Yarrowia lipolytica

Conditions for L-lactate oxidase synthesis by the yeast Yarrowia lipolytica were investigated. The enzyme was found to be synthesized during growth on L-lactate in the exponential growth phase. L-lactate oxidase synthesis was also observed on glucose after adaptation to stress conditions (oxidative or thermal stress) during the stationary growth phase after glucose consumption. The cells grown on L-lactate exhibited high levels of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase), which exceeded those of glucose-grown cells. Ultrastructurally, L-lactate-grown cells and the cells grown on glucose and adapted to various stress conditions were also found to be similar, with increased mitochondria, elevated number and size of peroxisomes, and formation of lipid and polyphosphate inclusions. In order to determine the intracellular localization of L-lactate oxidase, the cells were disintegrated by the lytic enzyme complex from Helix pomatia. Centrifugation of the homogenate in Percoll gradient resulted in the isolation of purified fractions of the native mitochondria and peroxisomes. L-lactate oxidase was shown to be localized in peroxisomes.     

Characterization and purification of carbon monoxide dehydrogenase from Methanosarcina barkeri.

Carbon monoxide-dependent production of H2, CO2, and CH4 was detected in crude cell extracts of acetate-grown Methanosarcina barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5 to 10 U/mg of protein). Carbon monoxide dehydrogenase activity was inhibited 85% by 10 microM KCN and was rapidly inactivated by O2. The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%). The native purified enzyme displayed a molecular weight of 232,000 and a two-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain nickel by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent Km of 5 mM for CO and a Vmax of 1,300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed.     

Purification and characterization of membrane-bound malate dehydrogenase from Acetobacter sp. SKU 14

Membrane-bound NAD(P)-independent malate dehydrogenase (EC 1.1.99.16) was purified to homogeneity from the membrane of thermotolerant Acetobacter sp. SKU 14, an isolate from Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Triton X-100 in the presence of 0.1 M KCl, and purified to homogeneity through steps of column chromatographies on DEAE-Sephadex A-50 and DEAE-Toyopearl in the presence of 0.1% Triton X-100. The purified enzyme showed a single protein band in both native-PAGE and SDS-PAGE. The enzyme was a homodimer with a molecular mass of 60 kDa subunit and had noncovalently bound FAD as the cofactor. The enzyme was stable over pH 5 and had its maximum activity at pH 11.0 when ferricyanide was used as an electron acceptor. The enzyme activity was elevated by the addition of ammonium ions. The substrate specificity was very strict to only L-malate, of which the apparent Km was 10 mM and over 20 compounds involving D-malate were not oxidized by the enzyme.     

Purification and some properties of carbon monoxide dehydrogenase from Acinetobacter sp. strain JC1 DSM 3803.

A brown carbon monoxide dehydrogenase from CO-autotrophically grown cells of Acinetobacter sp. strain JC1, which is unstable outside the cells, was purified 80-fold in seven steps to better than 95% homogeneity, with a yield of 44% in the presence of the stabilizing agents iodoacetamide (1 mM) and ammonium sulfate (100 mM). The final specific activity was 474 mumol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, NAD(P), flavin mononucleotide, flavin adenine dinucleotide, and ferricyanide were not reduced by the enzyme, but methylene blue, thionin, and dichlorophenolindophenol were reduced. The molecular weight of the native enzyme was determined to be 380,000. Sodium dodecyl sulfate-gel electrophoresis revealed at least three nonidentical subunits of molecular weights 16,000 (alpha), 34,000 (beta), and 85,000 (gamma). The purified enzyme contained particulate hydrogenase-like activity. Selenium did not stimulate carbon monoxide dehydrogenase activity. The isoelectic point of the native enzyme was found to be 5.8; the Km of CO was 150 microM. The enzyme was rapidly inactivated by methanol. One mole of native enzyme was found to contain 2 mol of each of flavin adenine dinucleotide and molybdenum and 8 mol each of nonheme iron and labile sulfide, which indicated that the enzyme was a molybdenum-containing iron-sulfur flavoprotein. The ratio of densities of each subunit after electrophoresis (alpha:beta:gamma = 1:2:6) and the number of each cofactor in the native enzyme suggest a alpha 2 beta 2 gamma 2 structure of the enzyme. The carbon monoxide dehydrogenase of Acinetobacter sp. strain JC1 was found to have no immunological relationship with enzymes of Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans.     

Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.     

Cloning of a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH): evidence for a second meningococcal L-LDH with different regulation.

We report the cloning of lldA, a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH). Escherichia coli contains a single L-LDH gene (lldD) in the lld operon (previously lct). E. coli grown in complex media does not have L-LDH activity, but the activity is induced by growth in defined medium with L-lactate as the carbon source. In contrast, meningococci contain at least one L-LDH in addition to the lldA gene product. These enzymes are active in meningococci grown in complex media and are not dependent on growth in L-lactate. The predicted amino acid sequence of lldA is homologous to that of E. coli lldD and of other prokaryotic and eukaryotic flavin mononucleotide-containing enzymes that catalyze the oxidation of L-lactate and other small alpha-hydroxy acids. A mutant with a deletion in lldA was found to have reduced L-LDH activity. However, this mutant was able to grow on L-lactate, indicating that a second L-LDH must exist. Activity of the lldA enzyme was affected by growth conditions, being increased by growth on a defined medium with either L-lactate or pyruvate as the carbon source. For meningococci grown on a complex medium, activity of the lldA enzyme was increased by growth on plates or in well-aerated broth. A second L-lactate-oxidizing activity was seen in bacteria grown in poorly aerated broth. Neisseria gonorrhoeae contains a homolog of lldA. As for meningococci, mutation of the gonococcal lldA reduced L-LDH activity but did not affect growth on L-lactate.     

Regulation of phosphoglycerate dehydrogenase levels and effect on serine synthesis in Escherichia coli K-12.

The level of phosphoglycerate dehydrogenase, the first enzyme in the biosynthetic pathway to serine and glycine, has been studied in Escherichia coli grown under different conditions. The enzyme level was not reduced by growth in a medium which contained the end products of the pathway, nor was it elevated when the growth rates was limited by the supply of serine. Elevation of phosphoglycerate dehydrogenase did not occur when charging of tRNA ser was inhibited by serine hydroxamate. However, phosphoglycerate dehydrogenase levels were subject to regulation. Elevated levels of enzyme activity were observed in merodiploids containing multiple copies of the serA gene, and lowered enzyme levels were found in cells grown on carbon sources other than glucose or when certain amino acids were present in the growth medium. The combined effect of these nutritional changes (carbon source and amino acids) reduced the level of phosphoglycerate dehydrogenase to 10 to 12% of that found in wild-type cells and to about 5% of the level in the merodiploids. By using antibody prepared against purified phosphoglycerate dehydrogenase we established that the decrease in enzyme activity reflected decreased amounts of enzyme protein. Constant intracellular concentrations of 3-phosphoglycerate and serine were found in cells with marked differences in phosphoglycerate dehydrogenase activity, indicating that end product inhibition of phosphoglycerate dehydrogenase activity, rather than the amount of the biosynthetic enzymes, is the major factor in regulating the intracellular concentration of serine.     

Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli.

Phosphorylated NADP+-isocitrate dehydrogenase (EC 1.1.1.42) has been purified to electrophoretic homogeneity from in vivo 32P-labeled Escherichia coli. The cells used as the source of phosphorylated enzyme were harvested 1 h after the addition of 5 mCi of [32P]orthophosphoric acid and 25 mM sodium acetate to cultures grown to early stationary phase on a low phosphate medium with limiting glucose. Double immunodiffusion and autoradiography demonstrated immunological identity between the 32P-labeled NADP+-isocitrate dehydrogenase and the enzyme isolated from glucose-grown E. coli. The phosphoenzyme had an apparent subunit molecular weight of 51,000 as determined by denaturing acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the radioactivity co-electrophoresed with NADP+-isocitrate dehydrogenase activity when purified enzyme was subjected to nondenaturing gel electrophoresis. [32P]Phosphoserine was identified following partial acid hydrolysis of the purified phosphoenzyme.     

Alcohol oxidation by Candida guilliermondii yeasts grown on hexadecanol

A number of enzyme systems involved in the first steps of hexadecane oxidation can be induced by hexadecanol, an intermediate product of hexadecane degradation. It has also been found that, in Candida guilliermondii cells and in their mitochondrial fraction, an oxidase system is induced when the cells are grown on hexadecanol. This system is similar to that in cells grown on hexadecane; it oxidises higher alcohols at a high rate and is not inhibited by the inhibitors of the man phosphorylating respiration chain. The membrane-bound alcohol dehydrogenase and aldehyde dehydrogenase activities resistant to pyrazole, an inhibitor of cytosol ethanol dehydrogenase, are induced together with the oxidase activity when the cells are grown on hexadecanol as well as on hexadecane. The oxidation of higher alcohols by whole cells is entirely inhibited by azide although their oxidation by mitochondria is resistant to the action of azide; apparently, azide inhibits the transport of alcohols into the cell.     

L-Lactate dehydrogenase from leaves of higher plants. Kinetics and regulation of the enzyme from lettuce (Lactuca sativa L).

1. L-Lactate dehydrogenase from lettuce (Lactuca sativa) leaves was purified to electrophoretic homogeneity by affinity chromatography. 2. In addition to its NAD(H)-dependent activity with L-lactate and pyruvate, the enzyme also catalyses the reduction of hydroxypyruvate and glyoxylate. The latter activities are not due to a contamination of the enzyme preparations with hydroxypyruvate reductase. 3. The enzyme shows allosteric properties that are markedly by the pH. 4. ATP is a potent inhibitor of the enzyme. The kinetic data suggest that the inhibition by ATP is competitive with respect to NADH at pH 7.0 and 6.2. The existence of regulatory binding sites for ATP and NADH is discussed. 5. Bivalent metal cations and fructose 6-phosphate relieve the ATP inhibition of the enzyme. 6. A function of leaf L-lactate dehydrogenase is proposed as a component of the systems regulating the cellular pH and/or controlling the concentration of reducing equivalents in the cytoplasm of leaf cells.     

D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum.

The gene encoding D-lactate dehydrogenase (D-lactate: NAD+ oxidoreductase, EC 1.1.1.28) of Lactobacillus plantarum has been sequenced, and expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5'-noncoding region of the gene was replaced with the tac promoter. Comparison of the sequence of D-lactate dehydrogenase with L-lactate dehydrogenases, including the L. plantarum L-lactate dehydrogenase, showed no significant homology. In contrast, the D-lactate dehydrogenase is homologous to E. coli D-3-phosphoglycerate dehydrogenase and Lactobacillus casei D-2-hydroxyisocaproate dehydrogenase. This indicates that D-lactate dehydrogenase is a member of a new family of 2-hydroxyacid dehydrogenases recently proposed, being distinct from L-lactate dehydrogenase and L-malate dehydrogenase, and strongly suggests that the new family consists of D-isomer-stereospecific enzymes. In the reductive reaction, the enzyme showed a broad substrate specificity, although pyruvate was the most favorable of all 2-ketocarboxylic acids tested. In particular, hydroxypyruvate is effectively reduced by the enzyme, the reaction rate, and Km value being comparable to those in the case of pyruvate, indicating that the enzyme has not only D-lactate dehydrogenase activity but also D-glycerate dehydrogenase activity. The conserved residues in this family appear to be the residues involved in the substrate binding and the catalytic reaction, and thus to be targets for site-directed mutagenesis.     

Post-proline dipeptidyl-aminopeptidase from synaptosomal membranes of guinea-pig brain. A possible role for this activity in the hydrolysis of His-ProNH2, arising from the action of synaptosomal membrane pyroglutamate aminopeptidase on thyroliberin

In this paper we report that while 55% of the total post-proline dipeptidyl-aminopeptidase activity in guinea-pig brain is associated with the soluble fraction of the cells, the remaining activity is widely distributed throughout the particulate fractions. A significant portion of this particulate activity is, however, associated with a synaptosomal membrane fraction. The specific activity of this enzyme rose as the synaptosomal membrane fraction was prepared from a synaptosomal fraction and had previously risen at the synaptosomal fraction was prepared from a postmitochondrial pellet. The synaptosomal membrane post-proline dipeptidyl-aminopeptidase was released from the membrane by treatment with Triton X-100 and partially purified by chromatography on Sephadex G-200. By contrast with the soluble enzyme the partially purified solubilised synaptosomal membrane post-proline dipeptidyl-aminopeptidase was not inhibited by 1.0 mM p-chloromercuribenzoate, 1.0 mM N-ethylmaleimide or 0.5 mM puromycin but was inhibited by 0.5 mM bacitracin. The partially purified solubilised enzyme was capable of releasing His-Pro from His-Pro-Val, His-Pro-Leu, His-Pro-Phe and His-Pro-Tyr and of releasing Gly-Pro from Gly-Pro-Ala but could not release Arg-Pro from Arg-Pro-Pro or from Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin). It was also unable to release Pro-Pro from Pro-Pro-Gly or Glp-Pro from Glp-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-MetNH2 (eledoisin). Using [Pro-3H]thyroliberin we show that the membrane-bound enzyme converts His-ProNH2, produced by the action of the synaptosomal membrane pyroglutamate aminopeptidase, to His-Pro thus competing with the spontaneous cyclisation of His-ProNH2 to His-Pro diketopiperazine. Purified preparations of synaptosomal membrane pyroglutamate aminopeptidase were used to generate His-ProNH2, which could then be converted to His-Pro by the presence of the partially purified synaptosomal membrane post-proline dipeptidyl-aminopeptidase. This preparation was free of contaminating post-proline cleaving endopeptidase, carboxypeptidase P, aminopeptidase P, prolyl carboxypeptidase or proline dipeptidase.     

L-Lactate dehydrogenase from Thermus caldophilus GK24, an extremely thermophilic bacterium. Desensitization to fructose 1,6-bisphosphate in the activated state by arginine-specific chemical modification and the N-terminal amino acid sequence

Heat-stable and fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) has been purified from an extremely thermophilic bacterium, Thermus caldophilus GK24 [Taguchi, H., Yamashita, M., Matsuzawa, H. and Ohta, T. (1982) J. Biochem. (Tokyo) 91, 1343-1348]. N-terminal sequence analysis of the first 34 amino acids of the enzyme indicates that the N-terminal arm region (first 1-20 residues) known for the vertebrate L-lactate dehydrogenases is completely missing in the T. caldophilus enzyme, while there is a high homology of sequence between the regions which are considered to be part of the NAD-binding domain. The C-terminal amino acid of the enzyme was phenylalanine. Analysis of the amino acid composition showed that T. caldophilus enzyme contained much more arginine and fewer lysine than other bacterial and vertebrate L-lactate dehydrogenases. On modification reaction with 2,3-butanedione in the presence of NADH and oxamate, an enhanced activity of the T. caldophilus L-lactate dehydrogenase was obtained independently of fructose 1,6-bisphosphate, and the modified enzyme was desensitized to fructose 1,6-bisphosphate. Amino acid analysis indicated that such a desensitization in the active state was caused by the modification of only one arginine residue per the enzyme subunit. Desensitization of the enzyme was inhibited in the presence of fructose 1,6-bisphosphate. A similar desensitization was observed using 1,2-cyclohexanedione instead of 2,3-butanedione. The enzyme was irreversibly modified with 2,3-butanedione and characterized. The irreversibly modified enzyme also showed an enhanced activity independently of fructose 1,6-bisphosphate, and its pyruvate saturation curve was similar to that of the native enzyme measured in the presence of fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate, which increases the thermostability of the native enzyme, did not affect that of the modified enzyme, while thermostability of the modified enzyme slightly decreased. Amino acid analysis indicated that only the arginine content was decreased by the modification. These results show that arginine residue(s) exist in the binding site for fructose 1,6-bisphosphate on the enzyme, and that the arginine residue(s) play some important role in the allosteric regulation of the enzyme activity.     

Overproduction of methanol dehydrogenase in glucose grown cells of a restricted RuMP type methylotroph

A restricted facultative methylotrophic RuMP type bacterium that can only utilize methanol and glucose has been found to possess a higher specific activity of methanol dehydrogenase during growth on glucose than during growth on methanol. The increased level of methanol dehydrogenase activity in glucose grown cells was the result of overproduction of the enzyme. In methanol grown cells 8% of the soluble protein consisted of methanol dehydrogenase, whereas in glucose grown cells the proportion amounted to 25%. The type of methanol dehydrogenase produced by this methylotroph could be separated from the crude extract and purified close to homogeneity in a one step procedure using cationic ion exchange chromatography. The enzyme is constitutive, and its level is determined by the growth rate.