Enzymatic phosphorylation of mannose by glucomannokinase from Mycobacterium phlei using inorganic polyphosphate.

Mannose-6-phosphate is an important phosphor-sugar, which is involved in many physiological functions and it is used to treat many diseases. Its production is however expensive since it requires costly substrate ATP as phosphorylation agent. This study has focused upon the direct synthesis of M6P by glucomannokinase using inorganic polyphosphate without involvement of ATP. The gene cloned for glucomannokinase has been sequenced from Mycobacterium phlei and it is transformed into Escherichia coli for expression. After purification involving affinity chromatography, a band of 30kDa corresponding to the enzyme has been isolated from induced crude supernatant. A total amount of 0.69mg/ml of enzyme has been successively obtained and the purity exceeds 90%. The kinetic assay studies show that this enzyme has more affinity towards polyphosphate and glucose than ATP and mannose respectively. The KM values of the enzyme for glucose, mannose, ATP and hexametaphosphate derived from experiments are 9.5, 203.7, 4.6, 1.7µM, respectively. The enzyme has shown a maximum production of mannose-6-phosphate at optimized conditions of pH 8.5, 25°C, poly(P)/mannose ratio 3:1 and in the presence of bivalent ion Mg2+. The results reveal that the glucomannokinase from Mycobacterium phlei suitable for further production of mannose-6-phosphate.

The YHS-Domain of an Adenylyl Cyclase from Mycobacterium phlei Is a Probable Copper-Sensor Module.

YHS-domains are small protein modules which have been proposed to bind transition-metal ions like the related TRASH-domains. They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases. Here we investigate a class IIIc adenylyl cyclase from Mycobacterium phlei which contains a C-terminal YHS-domain linked to the catalytic domain by a peptide of 8 amino acids. We expressed the isolated catalytic domain and the full-length enzyme in E. coli. The catalytic domain requires millimolar Mn2+ as a cofactor for efficient production of cAMP, is unaffected by low micromolar concentrations of Cu2+ and inhibited by concentrations higher than 10 µM. The full-length enzyme also requires Mn2+ in the absence of an activator. However, 1-10 µM Cu2+ stimulate the M. phlei adenylyl cyclase sixfold when assayed with Mn2+. With Mg2+ as the probable physiological cofactor of the adenylyl cyclase Cu2+ specifically switches the enzyme from an inactive to an active state. Other transition-metal ions do not elicit activity with Mg2+. We favor the view that the YHS-domain of M. phlei adenylyl cyclase acts as a sensor for copper ions and signals elevated levels of the transition-metal via cAMP. By analogy to TRASH-domains binding of Cu2+ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain.

Gene cloning and characterization of Mycobacterium phlei flavin reductase involved in dibenzothiophene desulfurization.

Mycobacterium phlei WU-F1 possesses the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range from 20 degrees C to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and a flavin reductase is essential in combination with these flavin-dependent monooxygenases. The flavin reductase gene (frm) of M. phlei WU-F1, which encodes a protein of 162 amino acid residues with a molecular weight of 17,177, was cloned and the deduced amino acid sequence shares approximately 30% identity with those of several flavin reductases in two protein-component monooxygenases. It was confirmed that the coexpression of frm with the DBT-desulfurization genes (bdsABC) from M. phlei WU-F1 was critical for high DBT-desulfurizing ability over a wide temperature range from 20 degrees C to 55 degrees C. The frm gene was overexpressed in Escherichia coli cells, and the enzyme (Frm) was purified to homogeneity from the recombinant cells. The purified Frm was found to be a 34-kDa homodimeric protein with a monomeric molecular mass of 17 kDa. Frm exhibited high flavin reductase activity over a wide temperature range, and in particular, the turnover rate for FMN reduction with NADH as the electron donor reached 564 s(-1) at 50 degrees C, which is one of the highest activities among all of the flavin reductases previously reported. Intriguingly, Frm also exhibited a high ferric reductase activity.

Identification and functional analysis of the genes encoding dibenzothiophene-desulfurizing enzymes from thermophilic bacteria.

Thermophilic bacteria Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1 desulfurize dibenzothiophene (DBT) and alkylated DBTs through specific cleavage of the carbon-sulfur bonds over a temperature range up to 52 degrees C. In order to identify and functionally analyze the DBT-desulfurization genes, the gene cluster containing bdsA, bdsB, and bdsC was cloned from B. subtilis WU-S2B. The nucleotide and amino acid sequences of bdsABC show homologies to those of the other known DBT-desulfurization genes and enzymes; e.g. a nucleotide sequence homology of 61.0% to dszABC of the mesophilic bacterium Rhodococcus sp. IGTS8 and 57.8% to tdsABC of the thermophilic bacterium Paenibacillus sp. A11-2. Deletion and subcloning analysis of bdsABC revealed that the gene products of bdsC, bdsA and bdsB oxidized DBT to DBT sulfone (DBTO(2)), converted DBTO(2) to 2'-hydroxybiphenyl-2-sulfinate (HBPSi), and desulfurized HBPSi to 2-hydroxybiphenyl (2-HBP), respectively. Resting cells of a recombinant Escherichia coli JM109 harboring bdsABC converted DBT to 2-HBP over a temperature range of 30-52 degrees C, indicating that the gene products of bdsABC were functional in the recombinant. The activities of DBT degradation at 50 degrees C and DBT desulfurization (2-HBP production) at 40 degrees C in resting cells of the recombinant were approximately five times and twice, respectively, as high as those in B. subtilis WU-S2B. The recombinant E. coli cells also degraded alkylated DBTs, such as 2,8-dimethylDBT and 4,6-dimethylDBT. The nucleotide sequences of B. subtilis WU-S2B bdsABC and the corresponding genes from M. phlei WU-F1 were found to be completely identical to each other although the strains are genetically different.

Partial characterization of a major autolysin from Mycobacterium phlei.

Autolytic enzyme profiles of fast- and slow-growing mycobacteria were examined using SDS-PAGE zymography with incorporated mycobacterial peptidoglycan sacculi as substrate. Each species tested (Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium kansasii) appeared to produce a different set of enzymes on the basis of differing number and molecular masses. A major autolysin from M. phlei was purified to apparent homogeneity by DEAE-cellulose chromatography, preparative gel electrophoresis and Mono Q FPLC. This enzyme had an estimated molecular mass of 38 kDa, an isoelectric point of 5.5 and a pH optimum of pH 7.5. Digestion of purified peptidoglycan by the enzyme resulted in the appearance of reducing sugars, suggesting that the 38 kDa autolysin is a beta-glycosidase. Partial internal amino acid sequence of the autolysin was determined and should facilitate identification, cloning and overexpression of the encoding gene.

Isolation, purification and characterization of intracellular calmodulin like protein (CALP) from Mycobacterium phlei.

A monomeric acidic protein of 14,000 Da with an isoelectric point of 4.5 was isolated from Mycobacterium phlei, which stained poorly with Coomassie brilliant blue. This protein showed retardation in mobility in SDS-PAGE upon treatment with calcium, similar to eukaryotic calmodulin proteins. Activation of cAMP phosphodiesterase and NAD kinase by this protein was observed. The CD spectral analysis indicated that the CALP has 52% of beta-conformation. The regular beta-conformation of the calmodulin like protein was shifted to 46% alpha-helical structure when calcium ions reacted with the protein, however, 42% of the CALP still retained its original beta-conformation. These observations indicated homology of this calcium binding protein with that of eukaryotic calmodulins in few structural and functional properties.

Polyamines exert regulatory control on mycobacterial transcription: a study using RNA polymerase from Mycobacterium phlei.

We have used purified RNA polymerase from Mycobacterium phlei to study the role of polyamines in mycobacterial transcription. Both initiation and elongation phases of the process were affected biphasically by polyamines. Interaction of polyamines with DNA template plays an important role in transcription modulation. Our studies emphasize that polyamines can exert a regulatory control on mycobacterial transcription.

Glutamine synthetase of Mycobacterium tuberculosis: extracellular release and characterization of its enzymatic activity.

We have investigated the activity and extracellular release of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] of Mycobacterium tuberculosis. The purified, homogeneous M. tuberculosis glutamine synthetase appears to consist of 12 most likely identical subunits of M(r) 58,000, arranged in two superimpose hexagons. In the catalysis of L-glutamine, the enzyme has an apparent Km for L-glutamate of approximately 3 mM at the pH optimum of 7.5. M. tuberculosis releases a large proportion (approximately 30%) of its total measurable enzyme activity into the culture medium, a feature that is highly specific for pathogenic mycobacteria. Immunogold electron microscopy revealed that M. tuberculosis also releases the enzyme into its phagosome in infected human monocytes. Two potentially important roles for glutamine synthase in the pathogenesis of M. tuberculosis infection are (i) the synthesis of L-glutamine, a major component of the cell wall of pathogenic but not nonpathogenic mycobacteria, and (ii) the modulation of the ammonia level in the M. tuberculosis phagosome, which may in turn influence phagosomal pH and phagosomelysosome fusion.

o-Succinylbenzoate: coenzyme A ligase, an enzyme involved in menaquinone (vitamin K2) biosynthesis, displays broad specificity.

o-Succinylbenzoate: coenzyme A ligase, an enzyme involved in menaquinone biosynthesis, was purified from Mycobacterium phlei and characterized with respect to isoelectric point, molecular weight, pH optimum, temperature optimum and kinetic data. The enzyme hydrolyses ATP to AMP. The substrate and cofactor specificity of the enzyme was tested with analogues of o-succinylbenzoic acid, different nucleotides, thiols and divalent cations. The enzyme appears to possess broad specificity for substrates and cofactors.

Sequence of proton abstraction and stereochemistry of the reaction catalyzed by naphthoate synthase, an enzyme involved in menaquinone (vitamin K2) biosynthesis.

The enzymic conversion of the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid (i.e. o-succinylbenzoic acid) to 1,4-dihydroxy-2-naphthoic acid is a cyclization reaction which is part of menaquinone (vitamin K2) biosynthesis. This conversion, which is probably a two-step process, was investigated using chirally labelled samples of the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid. To synthesize these, the following enzymes were employed: isocitrate: NADP+ oxidoreductase (EC 1.1.1.42), isocitrate glyoxylate-lyase (EC 4.1.3.1), 2-oxoglutarate dehydrogenase complex (which includes EC 1.2.4.2), 4-(2'-carboxyphenyl)-4-oxobutyrate synthase system and 4-(2'-carboxyphenyl)-4-oxobutyrate: CoA ligase. Isocitrate: NADP+ oxidoreductase was employed to generate the two enantiomeric samples of 2-oxoglutarate enantiotopically labelled at C-3. These samples were converted enzymically to succinate with retention of configuration at C-2 and C-3, and to 4-(2'-carboxyphenyl)-4-oxobutyric acid with retention of configuration at C-3. Isocitrate glyoxylate-lyase and isocitrate NADP+ oxidoreductase were employed to generate samples of 2-oxoglutarate enantiotopically tritiated at C-4 or at C-3 and C-4. The four variously labelled samples of 2-oxoglutarate were enzymically converted to the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid. The resulting variously labelled coenzyme A esters were incubated with naphthoate synthase to investigate the ring closure reaction. In the first step the 2HRe atom of the oxobutyric moiety of the coenzyme A ester is equilibrated with solvent protons in a fast and reversible reaction. Subsequently the 2HSi and 3HSi atoms are removed whereas the 3HRe atom becomes the proton at C-3 of 1,4-dihydroxy-2-naphthoic acid. The second step in this ring closure reaction is the rate-limiting step.

[Transamination processes in mycobacteria with different biological properties]. / Protsessy transaminirovaniia u mikobakterii s razlichnymi biologicheskimi svoistvami.

The activity of aspartate and alanine aminotransferases in mycobacteria of different specific affinity and the degree of resistance to antituberculous drugs were studied. Differences in alanine and aspartic acid transamination processes taking place in mycobacteria with different biological properties were demonstrated.

Physical and chemical characterization of glutamine synthetase purified from Mycobacterium phlei.

Glutamine synthetase (L-glutamate: ammonia ligase [ADP forming]) [EC 6.3.1.2] has been purified from a Gram-positive, acid-fast bacterium, Mycobacterium phlei, by simple procedures with 57% recovery. The enzyme resembled that from Mycobacterium smegmatis in the subunit size (56,000), molecular weight (670,000), amino acid composition, the amino acid sequence of the NH2-terminal, and the secondary structure. The enzyme activity was regulated by adenylylation of each subunit in the dodecameric molecule. M. phlei glutamine synthetase possesses two useful characteristics: high thermostability and resistance to protease digestion. The enzyme was not inactivated on exposure to 60 degrees C for 2 h or 37 degrees C for 72 h, or after incubation with 1% trypsin or chymotrypsin at 37 degrees C for 12 h, pH 7.8. With saturating substrate levels, the Arrhenius plot was nonlinear and concave downward with an intersection point at 45 degrees C, and the activation energies were calculated to be 3.2 and 9.6 cal/mol from the slopes. The specific activity of the highly adenylylated enzyme (E10.7) was remarkably lower than that of the slightly adenylylated enzyme (E2.5); however, both enzymes show similar profiles of the Arrhenius plot. These results indicate that the adenylylation of the enzyme does not affect its activation energies.

Influence of oxygen tension on the respiratory activity of Mycobacterium phlei.

Growth of Mycobacterium phlei under low oxygen tension resulted in specific activities two to twenty times lower for formate dehydrogenase, malate dehydrogenase, beta-hydroxybutyrate dehydrogenase, lactate oxidase and NADH dehydrogenase than when cultures were grown under high aeration. An increase in fumarate reductase and succinate dehydrogenase occurred with M. phlei grown under low oxygen tension. Malate: vitamin K dehydrogenase and glucose-6-phosphate dehydrogenase activity were not significantly affected by the oxygen tension used to grow the bacteria, and neither culture contained a lactate dehydrogenase. With growth of M. phlei in conditions of low oxygen tension, cytochrome a was not detected, but cytochrome b was prominent in membranes and cytochrome c was present in the soluble fraction.

4-(2'-Carboxyphenyl)-4-oxobutyryl coenzyme A ester, an intermediate in vitamin K2 (menaquinone) biosynthesis.

Enzyme preparations from Mycobacterium phlei, Escherichia coli and Galium mollugo cell suspension cultures were incubated in the presence of 4-(2'-carboxyphenyl)-4-oxobutyrate (i.e. o-succinylbenzoic acid, OSB, 1), ATP, coenzyme A and Mg2+. The main product isolated from the incubation mixture was 4-(2'-carboxyphenyl)-4-oxobutyryl coenzyme A ester (2) as determined by comparison with synthetic coenzyme A esters. Synthetic and enzymically formed 4-(2'-carboxyphenyl)-4-oxobutyryl coenzyme A ester (2) was shown to be enzymically converted to an intermediate in vitamin K2 biosynthesis viz. 1,4-dihydroxy-2-naphthoic acid (5). The enzymic formation of 2-(3'-Carboxypropionyl)benzoyl coenzyme A ester (3) and 4-(2'-carboxyphenyl)-4-oxobutyryl-di-coenzyme A ester (4) was also observed. They appeared in minor amounts, however. These esters were not convertible to 1,4-dihydroxy-2-naphthoic acid (5).

NADP-specific isocitrate dehydrogenase of Mycobacterium phlei ATCC 354: purification and characterization.

NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) from Mycobacterium phlei ATCC 354 was purified to homogeneity by ammonium sulphate fractionation, followed by DEAE cellulose and Sephadex G-200 chromatography. The pH optimum of the enzyme was 8.5. The Km values for isocitrate and NADP were 74 and 53 microM, respectively. Mn2+ was essential for enzyme activity. The enzyme lost all activity on incubation at 70 degrees C for 15 min; isocitrate and NADP protected against this thermal inactivation. p-Chloromercuribenzoate inhibited the enzyme; pre-incubation of enzyme with isocitrate + Mn2+ prevented this inhibition. The purified enzyme showed concerted inhibition by glyoxylate + oxaloacetate and was inhibited by oxalomalate.

Characterization of mycobacterial immunoprecipitates by selective staining of enzymes.

Immune precipitation patterns of Mycobacterium intracellulare, M. phlei and M. smegmatis were analysed by selective enzyme staining procedures in order to characterize individual mycobacterial antigens. Enzyme activity was shown in eight precipitinogens of M. intracellulare, seven of M. phlei, and six of M. smegmatis. The identification of mycobacterial precipitinogens as enzymes is important since only a few mycobacterial antigens have been functionally characterized.

Metabolic studies on mycobacteria. III. Demonstration of key enzymes of TCA cycle in M. leprae.

Cell free extracts of armadillo derived M. leprae, M. phlei, M. smegmatis and normal armadillo liver were analysed for the two key enzymes of TCA cycle. Aconitase activity was assayed in the presence of inhibitor fluorocitrate and it was observed that cell free extracts from cultivable mycobacteria as well as aramadillo derived M. leprae had this enzyme activity and 66-82% of this activity was inhibited by 0.1 mM fluorocitrate. 74% of M. leprae derived enzyme activity was inhibited by fluorocitrate in contrast with armadillo derived enzyme which was only 29% inhibited by fluorocitrate. PAGE separation of cell free extracts and staining for Isocitrate dehydrogenase (ICD) activity showed that an additional bond of ICD activity was demonstrable in the cell free extracts of armadillo derived M. leprae and this was NADP dependent. The mobility (ef) of this band of activity was in the same range as ICD from cultivable mycobacteria and much lower than ICD from normal armadillo liver. From this study and from the previously reported work, it is concluded that like other mycobacteria TCA cycle is operative in M. leprae.

Solubilization, purification, and characterization of succinate dehydrogenase from membranes of Mycobacterium phlei.

Succinate dehydrogenase (SDH) was solubilized from membranes of Mycobacterium phlei by Triton X-100 with a recovery of about 90%. The solubilized SDH was purified about 90-fold by Sephacryl S-300, DEAE-cellulose, hydroxylapatite, and isoelectric focusing in the presence of Triton X-100 with a 20% recovery. SDH was homogeneous, as determined by polyacrylamide gel electrophoresis in nondenaturing gels containing Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme revealed two subunits with molecular weights of 62,000 and 26,000. SDH is a flavoprotein containing 1 mol of flavin adenine dinucleotide, 7 to 8 mol of nonheme iron, and 7 to 8 mol of acid-labile sulfide per mol of protein. Using phenazine methosulfate and 2,6-dichloroindophenol as electron acceptors, the enzyme had an apparent Km of 0.12 mM succinate. SDH exhibited a sigmoidal relationship of rate to succinate concentration, indicating cooperativity. The enzyme was competitively inhibited by fumarate with a Ki of 0.15 mM. In the absence of Triton X-100, the enzyme aggregated, retained 50% of the activity, and could be resolubilized with Triton X-100 with full restoration of activity. Cardiolipin had no effect on the enzyme activity in the absence of Triton X-100, but it stimulated the activity by about 30% in the presence of 0.1% Triton X-100 in the assay mixture. Menaquinone-9(2H), isolated from M. phlei, had no effect on the enzyme activity either in the presence or absence of Triton X-100.

Purification and characterization of dihydrofolate reductase from Mycobacterium phlei.

The dihydrofolate reductase from Mycobacterium phlei was purified and characterized; it has an Mr of 15 000 and a pI of 4.8. It is competitively inhibited by both methotrexate and trimethoprim, although the affinity is less than for other bacterial dihydrofolate reductases.

Isoenzyme patterns of mycobacteria. I. Factors influencing LDH isoenzymes of mycobacteria.

Cell free extracts of a fast growing mycobacterium (M. phlei) and a slow growing mycobacterium (M. tuberculosis H37Ra) were analysed for lactate dehydrogenase (LDH) isoenzymes under different experimental conditions. It was observed that growth of M. phlei when taken from Lowenstein Jensen (LJ) as well as Sauton's medium showed identical band but for (M. tuberculosis H37Ra the number of bands observed were less when grown on LJ-medium. There was no difference in LDH isoenzyme patterns when the mycobacteria were incubated at 30 degrees C and 37 degrees C and under different pH conditions (6.2-8.2). Actively growing cultures of both the species showed distinct LDH isoenzyme patterns whereas the activity and bands became indistinct in old cultures. The LDH bands from lyophilized growth studied resembled to those of fresh growth. The treatment of growth with 1M NaOH for one hour resulted in marked diminution of LDH activity. Sonication with wet growth weight of 0.5 gm per ml of distilled water was found to give clearer bands as compared to phosphate buffer. No loss of LDH isoenzymes activity was noticed after storing the extracts at -80 degrees C for one month, treating to 58 degrees C for one hour or freezing and thawing for 2 times whereas these isoenzymes were quite unstable at other storage temperatures. Increasing the staining time was found helpful in getting clearer bands when activity was low. It is concluded that the factors studied have important bearing on LDH isoenzyme patterns of mycobacteria and must be kept in mind while studying the LDH zymograms for any taxonomic identification of mycobacteria or for studying the metabolic role. These are important both for sensitivity and reproducibility of LDH zymograms.