Identification and isolation of glucose dehydrogenase genes of Bacillus megaterium M1286 and their expression in Escherichia coli

A gene encoding glucose dehydrogenase of Bacillus megaterium M1286 was isolated from a lambda-EMBL3 phage library. It is transcribed and translated in cells of the heterologous organism Escherichia coli by own control regions. The gene is located on a 1126-bp HindIII fragment. Its nucleotide sequence contains 220 bp in the 5' non-coding region, 783 bp in the coding region and 123 bp in the 3' non-coding region. The amino acid sequence, as deduced from the coding region, consists of 261 amino acids and is different from the known protein sequence of glucose dehydrogenase from B. megaterium M1286. [Jany, K. D., Ulmer, W., Fr?schle, M. & Pfleiderer, G. (1984) FEBS Lett. 165, 6-10]. By using this gene as a hybridization probe a second glucose dehydrogenase gene was isolated, which was also directly expressed in E. coli. Additionally a DNA region with extended sequence homology to the hybridization probe was identified. This work indicates the existence of at least two independent glucose dehydrogenase genes in B. megaterium M1286. Homologies in the primary structures of the two different glucose dehydrogenases of B. megaterium M1286 and of the corresponding Bacillus subtilis enzyme are discussed.     

Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium.

Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.     

Proline iminopeptidase from Bacillus megaterium: purification and characterization

Proline iminopeptidase [EC 3.4.11.5] was purified about 1,700-fold from cell free extract of Bacillus megaterium by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose and hydroxyapatite, and gel filtration on Toyopearl FW-55. The purified enzyme still contained a minor contaminant as judged by disc gel electrophoresis. The enzyme was most active at pH 7.0 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminal. The enzyme was completely inactivated by p-chloromercuribenzoate (PCMB), but was not inhibited by metal chelators, diisopropylphosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by adding 2-mercaptoethanol. From this result and the chromatographic profile on PCMB-T-Sepharose, the enzyme seems to be a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0. The molecular weight of the enzyme was estimated to be 58,000 by gel filtration on Toyopearl and 60,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is a monomer.     

Enzymatic properties of isozymes and variants of glucose dehydrogenase from Bacillus megaterium

Three glucose dehydrogenases (GlcDH) from Bacillus megaterium, GlcDH-I, GlcDH-II and GlcDH-IWG3, were purified from Escherichia coli cells harboring one of the hybrid plasmids, pGDK1, pGDK2 and pGDA3, respectively, pGDK1 and pGDK2 contain two isozyme genes, gdhI and gdhII, respectively, from B. megaterium IAM 1030 and pGDA3 contains an isozyme gene from B. megaterium IWG3; GlcDH-IWG3 is a variant of GlcDH-I. GlcDH-I and GlcDH-II have similar pH/activity profiles and the profile for GlcDH-IWG3 is identical to that of GlcDH-I. The pH/stability profiles of these enzymes show that GlcDH-IWG3 is the most stable enzyme in the acidic region, while GlcDH-II is the most stable in the alkaline region, and GlcDH-I is the most unstable throughout the entire pH range examined. As for thermostability, GlcDH-II is the most resistant against heat inactivation at pH 6.5. The values of the first-order rate constant for heat inactivation at 50 degrees C are 0.27 min-1, 0.05 min-1 and 0.11 min-1 for GlcDH-I, GlcDH-II and GlcDH-IWG3, respectively. Kinetic studies show that these enzymes have similar kinetic constant values except that there are some differences in Kia for NAD(P) and Ka (the limiting Michaelis constant) for NAD; the values of the ratio of Kia for NAD and NADP are 11,340 and 8.7 for GlcDH-I, GlcDH-II and GlcDH-IWG3, respectively. GlcDH-I and GlcDH-IWG3 have very similar substrate specificities and GlcDH-II has a slightly higher specificity for D-glucose and 2-deoxy-D-glucose than the others. The results are discussed on the basis of the amino acid substitutions between the enzymes.     

Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030.

Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.     

Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli. I: Purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium

Escherichia coli containing the Bacillus subtilis glucose dehydrogenase gene on a plasmid (prL7) was used to produce the enzyme in high quantities. Gluc-DH-S was purified from the cell extract by (NH4)2SO4-precipitation, ion-exchange chromatography and Triazine-dye chromatography to a specific activity of 375 U/mg. The enzyme was apparently homogenous on SDS-PAGE with a subunit molecular mass of 31.5 kDa. Investigation of Gluc-DH-S was performed for comparison with the corresponding properties of Gluc-DH-M. The limiting Michaelis constant at pH 8.0 for NAD+ is Ka = 0.11 mM and for D-glucose Kb = 8.7 mM. The dissociation constant for NAD+ is Kia = 17.1 mM. Similar to Gluc-DH-M, Gluc-DH-S is inactivated by dissociation under weak alkaline conditions at pH 9.0. Complete reactivation is attained by readjustment to pH 6.5. Ultraviolet absorption, fluorescence and CD-spectra of native Gluc-DH-S, as well as fluorescence- and CD-backbone-spectra of the dissociated enzyme were nearly identical to the corresponding spectra of Gluc-DH-M. The aromatic CD-spectrum of dissociated Gluc-DH-S was different, representing a residual ellipticity of tryptophyl moieties in the 290-310 nm region. Density gradient centrifugation proved that this behaviour is due to the formation of inactive dimers in equilibrium with monomers after dissociation. In comparison to Gluc-DH-M, the kinetics of inactivation as well as the time-dependent change of fluorescence intensity at pH 9.0 of Gluc-DH-S showed a higher velocity and a changed course of the dissociation process.     

Improved purification and properties of glucose dehydrogenase from Bacillus subtilis

Glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was purified about 5240-fold, using an aqueous two-phase system and triazine-dye affinity chromatography. The specific activity of the purified preparation was about 460 units/mg of protein with a final recovery of enzyme activity of about 75%. The affinity column could be regenerated and reused again several times. The purified enzyme appeared to be homogeneous when analyzed both on SDS-PAGE and native PAGE. The protein band on native PAGE coincided with the activity stain. ATP acts apparently as a competitive inhibitor for this enzyme with respect to NAD and protects the enzyme from dissociation into partially inactive dimers. In the absence of either glycerol or ATP, the enzyme dissociates into partially inactive dimers.     

Pterin deaminase from Bacillus megaterium. Purification and properties.

A pterin deaminase catalyzing the hydrolytic deamination of various pteridines was found in the bacterium, Bacillus megaterium, and partially purified from bacterial extract. The specific activity was raised 90-fold over that of the crude extract. The pH optimum is around 7.3, and the Km value for 6-carboxypterin is 1.3 mM. The molecular weight of the enzyme was estimated by gel filtration to be about 110,000. The enzyme deaminated pterin, 6-carboxypterin, biopterin, 6-methylpterin, 7-methylpterin, xanthopterin, 6-hydroxymethylpterin, sepiapterin, isosepiapterin, folic acid, and 6,7-dimethylpterin to their corresponding lumazines, whereas guanine, 7-carboxypterin, leucopterin, isoxanthopterin, and 6-methylisoxanthopterin did not serve as substrates. The enzyme was inhibited by PCMB and 8-azaguanine.     

Primary structure, expression in Escherichia coli, and properties of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from Bacillus megaterium.

A Bacillus megaterium DNA fragment encoding S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity was subcloned and sequenced. The encoded polypeptide showed more than 43.5% strict homology to Pseudomonas denitrificans SUMT (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet, and B. Cameron, J. Bacteriol. 171:4222-4231, 1989). The B. megaterium polypeptide was overexpressed in Escherichia coli, partially purified, and shown to exhibit, like P. denitrificans SUMT, substrate inhibition at uroporphyrinogen III concentrations above 0.5 microM, suggesting a common regulation for aerobic cobalamin-producing organisms.     

Isolation and properties of membranes from Bacillus megaterium spores.

Membranes from dormant and heat-activated spores of Bacillus megaterium QM B1551 were isolated and purified by gentle lysis procedures followed by differential and sucrose density gradient centrifugations. The purified membranes were enriched for inner membranes and were characterized by their density and content of proteins, phospholipids, enzymes, cytochromes, and carotenoids. These purified spore membranes could be used to investigate their role in the triggering of germination.     

Crystal structure of glucose dehydrogenase from Bacillus megaterium IWG3 at 1.7 A resolution

The crystal structure of glucose dehydrogenase (GlcDH) from Bacillus megaterium IWG3 has been determined to an R-factor of 17.9% at 1.7 A resolution. The enzyme consists of four identical subunits, which are similar to those of other short-chain reductases/dehydrogenases (SDRs) in their overall folding and subunit architecture, although cofactor binding sites and subunit interactions differ. Whereas a pair of basic residues is well conserved among NADP(+)-preferring SDRs, only Arg39 was found around the adenine ribose moiety of GlcDH. This suggests that one basic amino acid is enough to determine the coenzyme specificity. The four subunits are interrelated by three mutually perpendicular diad axes (P, Q, and R). While subunit interactions through the P-axis for GlcDH are not so different from those of the other SDRs, those through the Q-axis differ significantly. GlcDH was found to have weaker hydrophobic interactions in the Q-interface. Moreover, GlcDH lacks the salt bridge that stabilizes the subunit interaction in the Q-interface in the other SDRs. Hydrogen bonds between Q-axis related subunits are also less common than in the other SDRs. The GlcDH tetramer dissociates into inactive monomers at pH 9.0, which can be attributed mainly to the weakness of the Q-axis interface.     

Enolase from spores and cells of Bacillus megaterium: two-step purification of the enzyme and some of its properties.

A simple two-step procedure for purification of enolase from germinated spores or vegetative cells of Bacillus megaterium is described. The procedure resulted in a 1,200-fold purification with production of homogeneous enzyme in approximately 75% yield; the enzymes from spores and cells seemed identical. The molecular weight of the native enzyme was 335,000, with a subunit molecular weight of 42,000. The enzyme required Mg2+ and was inhibited by ethylenediaminetetraacetic acid and fluoride ions. The Michaelis constants for 2-phosphoglyceric acid and Mg2+ were 7.1 X 10(-4) and 4.7 X 10(-4) M, respectively.     

The purification and characterization of a DNA nicking-closing enzyme from Bacillus megaterium.

Although several eucaryote DNA nicking--closing (N--C) enzymes have been characterized, only the Escherichia coli enzyme has been extensively studied amongst procaryotes. The latter enzyme is distinctly different from the eucaryotic enzymes and we have therefore purified the N--C enzyme from Bacillus megaterium to determine if procaryotes form a distinctive class of N--C enzymes. The purified B. megaterium N--C enzyme has a molecular weight of 120,000, only partly relaxes negative supercoils, does not affect positive supercoils, requires Mg2+, and is inhibited by 0.2 M KCl. The enzyme is also inhibited by 1 mM nalidixic or oxolinic acids but unaffected by novobiocin. A crude N--C enzyme preparation from Micrococcus luteus shows very similar properties.     

Correlation between spore structure and spore properties in Bacillus megaterium

The spores of six strains of Bacillus megaterium were divided into two distinct groups on the basis of germination. Three of the strains germinated in a mixture of l-alanine and inosine (AL type spores), and three strains germinated in a mixture of glucose and potassium nitrate (GN type spores); recriprocal germination in the respective solutions did not occur. The AL spores and the GN spores were morphologically distinct. Other differences between the two spore groups included germination inhibition characteristics, dipicolinic acid content, hexosamine content, phosphorus and magnesium content, spore coat features, ion exchange properties, and heat resistance. A correlation appears to exist between spore morphology and certain other spore properties in strains of B. megaterium.