13C and 15N isotope effects for conversion of L-dihydroorotate to N-carbamyl-L-aspartate using dihydroorotase from hamster and Bacillus caldolyticus

In the pyrimidine biosynthetic pathway, N-carbamyl-L-aspartate (CA-asp) is converted to L-dihydroorotate (DHO) by dihydroorotase (DHOase). The mechanism of this important reaction was probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and 45 degrees C for hamster DHOase; 37, 50, and 60 degrees C for Bacillus caldolyticus), and the product CA-asp was purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the beta-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary and secondary isotope effects for the reaction catalyzed by DHOase showed little variation between enzymes or temperatures, the primary 13C and 15N isotope effects being approximately 1% on average, while the secondary 13C isotope effect is negligible or very slightly normal (>1.0000). These data indicate that the chemistry is at least partially rate-limiting while the secondary isotope effects suggest that the transition state may have lost some bending and torsional modes leading to a slight lessening of bond stiffness at the carbonyl carbon of the amide of CA-asp. The equilibrium isotope effects for DHO --> CA-asp have also been measured (secondary 13K(eq) = 1.0028 +/- 0.0002, primary 13K(eq) = 1.0053 +/- 0.0003, primary 15K(eq) = 1.0027 +/- 0.0003). Using these equilibrium isotope effects, the kinetic isotope effects for the physiological reaction (CA-asp --> DHO) have been calculated. These values indicate that the carbon of the amide group is more stiffly bonded in DHO while the slightly lesser, but still normal, values of the primary kinetic isotope effect show that the chemistry remains at least partially rate-limiting for the physiological reaction. It appears that the ring opening and closing is the slow step of the reaction.     

Ribosomal proteins and DNA-binding protein II from the extreme thermophile Bacillus caldolyticus

Crystallographic studies, presently on ribosomal and DNA-binding proteins from the moderate thermophile Bacillus stearothermophilus, can be expected to benefit from the use of even more stable proteins from extreme thermophiles. Bacillus caldolyticus, which is able to grow in the temperature range of 70-80 degrees C, appears to be a suitable candidate. We have compared the two bacilli using two criteria: the two-dimensional gel patterns of ribosomal proteins and the properties of DNA-binding protein II. The latter protein is ubiquitous in the eubacterial kingdom and can be purified in large quantities. B. caldolyticus can be grown at 75 degrees C in continuous culture with a generation time of 45-60 min. The yield of ribosomes compares favorably with that of B. stearothermophilus. The gel patterns of the ribosomal proteins are very similar but several differences, in particular among the 50S proteins, are observed. The N-terminal amino-acid sequence of the DNA-binding protein differs in 3 positions (out of 39) from B. stearothermophilus and the protein shows an increased resistance to thermal denaturation. Tetragonal and monoclinic crystals of DNA-binding protein II have been obtained which are suitable for X-ray studies and the diffraction patterns of the two crystal forms are shown.     

Separation and partial purification of extracellular amylase and protease from Bacillus caldolyticus

Summary The extracellular amylase and protease from Bacillus caldolyticus can be concentrated by ammonium sulfate precipitation after growth on either solid or in liquid media containing starch, glucose, and brain-heart infusion. Using the Diaflo ultrafiltration system with membranes of various permeability, the enzymes could be separated from each other by extensive flushing with buffer. Best results were obtained with the 50–70% ammonium sulfate fraction as starting material, yielding 72% of the total amylase activity in the low molecular weight fraction (UM-10 fraction: 10000–30000), while 54 and 25% respectively of the protease were retained in the two high molecular weight fractions (50000–100000, and more than 100000). Similar results were obtained with the 20–50% ammonium sulfate fraction, while the fraction of 0–20% saturation contained a low molecular weight protease. The native amylase seems to consist of a number of sub-units, which after extensive flushing accumulate in the fraction with an approximate molecular weight between 10000 and 30000. The enzyme could also be precipitated from cell-free liquid media with ammonium sulfate, followed by separation and purification on ultra-filtration cells. According to the specific activity of the UM-10 fractions a 400-fold purification was obtained compared to the amylase activity of the cell-free medium.Direct concentration and separation from liquid media, omitting ammonium sulfate treatment, was also found to be possible, although prolonged flushing with buffer was necessary to obtain satisfactory separation.During purification from the ammonium sulfate fractions, amylase activity was found to decrease but could be restored by Ca-ions. At 70°C, a final concentration of 0.5 mM CaCl₂, was sufficient for full restoration, while three times that amount was necessary at 80°C. Determination of the K _m-values for Ca at different temperatures resulted in an asymptotically increasing curve at temperatures beyond 75°C. Addition of Ca had a pronounced effect on the stability of the amylase at 80°C but not at 90°C. Protease activity and stability was not affected by Ca-ions.     

Complementarity of regulation for the two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile

Chromatophores from Rhodopseudomonas capsulata cells grown semiaerobically in the dark oxidize NADH, succinate, and dichlorophenolindophenol. In the presence of N₃^? these activities are inhibited, but light induces oxidation of dichlorophenolindophenol with O₂ as a terminal electron acceptor. Cyanide also inhibits electron transport but much higher concentrations are required to inhibit the photooxidation than the dark oxidation. The photooxidation was studied in a mutant strain of Rhodopseudomonas capsulata (YIV) which cannot grow anaerobically in the light, but similarly to the wild type, grows in the presence of oxygen. Chromatophores from YIV mutant catalyze photophosphorylation and dark oxidation activities with the same properties as those of the wild type. However, the rate of photooxidation in the mutant is only one-third that of the wild type. Based on the differential inhibitor sensitivity and on the mutation it is suggested that the photooxidase is different from the two respiratory oxidases and that this photooxidation activity might be essential for growth of the cells under anaerobic conditions in the light.     

Two regulatory isozymes of glutamine synthetase from Bacillus caldolyticus, an extreme thermophile.

Two glutamine synthetases (EC 6.3.1.2) have been purified to homogeneity from $\text{B. caldolyticus}$ strain YTP, grown at 70° on a minimal, defined medium. The enzymes are virtually identical in size and molecular weight (12 sub-units of MW 50,000), but differ in their isoelectric points, electrophoretic mobility, net charge, inherent thermal stability, affinity for substrates, and activity responses to metal ions and pH. Of primary interest is the observation that the more acidic form (pI = 5.2), E_I, is strongly feedback-regulated by certain amino acids derived from glutamine (Gly, L-Ala, L- and D-ser) but not by L-Glu or AMP, whereas the less acidic form (pI = 5.5), E_II, is inhibited most strongly by L-Gln and AMP, but not by the above amino acids. Both enzymes are inhibited strongly by ADP, CTP, NAD, glucosamine-6-P, less strongly by nucleotide diphosphates and L-Trp, and are activated by nucleotide monophosphates other than AMP. These results suggest that overall regulation of glutamine synthetase by the full spectrum of end product metabolites derived from L-Gln is accomplished by regulatory isozymes in this extremely thermophilic organism.     

Effect of Temperature on the Fatty Acid Composition of the Extreme Thermophiles, Bacillus caldolyticus and Bacillus caldotenax

Gas chromatographic analysis of extracts from Bacillus caldolyticus and Bacillus caldotenax grown at 60, 70, or 80 C showed that both contain branched-chain fatty acids as major constituents at all temperatures tested. With increasing temperature, a decrease of i-C15 and an increase of i-C17 fatty acids were observed in both strains, as well as a decrease of i-C16 fatty acids corresponding to an increase of n-C16 fatty acids. The most obvious difference was that the shifts observed with B. caldolyticus occurred mainly upon raising the temperature to 10 C above, and in B. caldotenax upon lowering the temperature to 20 C below, the optimal growth temperature.     

Correlation between the fatty acid composition and the activity of extracellular enzymes from Bacillus caldolyticus

Summary Bacillus caldolyticus, grown at 70°C, produces a highly active extracellular amylase and protease. Both enzymes are formed either within the membrane, or at its inner surface. The activity of both extracellular enzymes was found to decline drastically when brain-heart infusion was omitted from the medium. A simultaneous increase of both enzymes inside the cell was observed. The shifting in extra- and intra-cellular activity was caused by changes in membrane composition due to the increase of anteiso-odd and n-even, and the decrease of iso-odd fatty acids. Membrane composition and enzymic activity could be influenced by the addition of either leucine or iso-leucine as precursors for the synthesis of branched-chain fatty acids: In presence of leucine the anteiso-odd and n-even fatty acids returned to their normal level, while the iso-odd fatty acids increased. Simultaneously the extracellular protease activity increased, and the intracellular activity declined. Growth in amylose-medium supplied with leucine lead to a decrease of both the intra- and extracellular amylase, and changes in the fatty acid composition of the membrane which could not be restored by transfer of the organism to complete media. Addition of iso-leucine first lead to a sharp decrease of extracellular protease and a drastic increase of intracellular protease activity, accompanied by an increase of anteiso-odd and n-even fatty acids, and a decrease of iso-odd compounds. After the second growth in presence of iso-leucine the intra- and extra-cellular protease activity was reversed, and thus showed a return to the starting situation. The reversal is accompanied by the preferential incorporation of fatty acids with a higher melting point into the membrane. Extracellular amylase activity was found to increase after the first growth with iso-leucine, and to decline sharply after the second culture with iso-leucine, together with a very high intracellular amylase activity at that point. Extra- and intra-cellular amylase activity both declined upon growth in complete medium, while the fatty acid distribution remained different from the initial composition.     

Intermediates in guanidine-HC1 unfolding of glutamine synthetase from the extreme thermophile, Bacillus caldolyticus.

Glutamine synthetase is expressed in Bacillus caldolyticus as two isoforms that differ in physico-chemical and regulatory properties. Biphasic kinetics of thermal denaturation of E-I and E-II (Merkler, D.J., et al (1987) Biochemistry 26, 7805), suggested the formation of intermediates. CD spectral changes of E-II induced by guanidine-HC1 clearly indicate a three-state pathway for unfolding (N----I----D). Refolding of E-II from 6 M GuHCl led to only 15% recovery of activity, compared to greater than or equal to 90% with E-I.     

Two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile. Isolation, physicochemical and kinetic properties

Two distinctly different glutamine synthetase enzymes (EI and EII) have been isolated from the extreme thermophile Bacillus caldolyticus, grown on chemically defined medium at 70 degrees C. Purification to homogeneity mainly involves affinity chromatography and heat treatment with substrate protection. Biosynthesis of total enzyme activity can be repressed by at least 8-fold by high ammonia, with synthesis of EI being repressed more strongly than EII. A variety of chemical and biochemical tests failed to provide evidence for regulation of EI or EII by covalent modification, e.g. proteolysis, phosphorylation, or adenylylation. Neither of the thermophiic enzymes will cross-react with antibodies for the Escherichia coli or Bacillus subtilis glutamine synthetases. Both enzymes are composed of 12 subunits, each approximately 51,000 daltons. However, EI and EII differ significantly in their amino acid composition, isoelectric points (5.2 and 5.5, respectively), rates of migration on polyacrylamide electrophoresis gels at pH 6.8, and kinetic properties, EI is more active with Mg(II) than with Mn(II), but EII is more active with Mn(II) than Mg(II). Cd(II) activates EII more than EI, and only EI shows activity with Co(II). For both enzymes, the Mn(II)-stimulated activity is optimal at pH 6.0 to 6.5, with Mn(II)/ATP = 1.0, but the pH optimum with Mg(II) is near pH 7.5, however, with a ratio of Mg(II)/ATP > 2. Substrate Km values at 70 degrees C differ for EI versus EII but are quite comparable to those seen for mesophilic glutamine synthetases. Studies with structural analogs of substrates indicate that active site specificity is maintained at extreme temperatures: substitution of alpha-OH for alpha-HN2 is allowed, but unfavorable changes occur upon substitution of methyl groups for the alpha-H or onto the alpha-NH2 of L-Glu, and for D-Glu or L-Asp. EII is almost absdolutely specific for ATP, but EI can also use ITP, GTP, and UTP as substrates to some extent. The divalent metal ion that is present can affect both specificity for analogs and substrate Km values. Kinetic binding plots (v versus [S]) are biphasic for NH3 and L-Glu with the more active forms of each enzyme, EI-Mg and EII-Mn, respectively; but no positive cooperativity is observed. ATP binding is strictly hyperbolic, in contrast to the positive cooperativity previously observed with other Bacillus sp. enzymes. For purified EI and EII, Arrhenius plots are nonlinear with Mn(II) or Mg(II), exhibiting slope changes in the range of 55-65 degrees C; however, for EI-EII mixtures in crude cell extracts these plots are nearly linear.     

Thermal stability and atomic-resolution crystal structure of the Bacillus caldolyticus cold shock protein

The bacterial cold shock proteins are small compact beta-barrel proteins without disulfide bonds, cis-proline residues or tightly bound cofactors. Bc-Csp, the cold shock protein from the thermophile Bacillus caldolyticus shows a twofold increase in the free energy of stabilization relative to its homolog Bs-CspB from the mesophile Bacillus subtilis, although the two proteins differ by only 12 out of 67 amino acid residues. This pair of cold shock proteins thus represents a good system to study the atomic determinants of protein thermostability. Bs-CspB and Bc-Csp both unfold reversibly in cooperative transitions with T(M) values of 49.0 degrees C and 77.3 degrees C, respectively, at pH 7.0. Addition of 0.5 M salt stabilizes Bs-CspB but destabilizes Bc-Csp. To understand these differences at the structural level, the crystal structure of Bc-Csp was determined at 1.17 A resolution and refined to R=12.5% (R(free)=17.9%). The molecular structures of Bc-Csp and Bs-CspB are virtually identical in the central beta-sheet and in the binding region for nucleic acids. Significant differences are found in the distribution of surface charges including a sodium ion binding site present in Bc-Csp, which was not observed in the crystal structure of the Bs-CspB. Electrostatic interactions are overall favorable for Bc-Csp, but unfavorable for Bs-CspB. They provide the major source for the increased thermostability of Bc-Csp. This can be explained based on the atomic-resolution crystal structure of Bc-Csp. It identifies a number of potentially stabilizing ionic interactions including a cation-binding site and reveals significant changes in the electrostatic surface potential.     

Variability of the molecular size of extracellular amylase produced by intact cells and protoplasts of Bacillus caldolyticus

One of the typical properties of the extracellular amylase produced by Bacillus caldolyticus is the tendency to disintegrate into subunits with a molecular weight (MW) of less than 10,000, when the enzyme is subjected to ultrafiltration. Disintegration is due to a loss of Ca-ions, leading to nonactive subunits. Activity can be fully restroed by addition of Ca-ions. Reactivation occurs also spontaneously if the low MW fraction is stored in glassware. Comparative ultrafiltration experiments with the subunit fraction with or without a supply of Ca revealed that in presence of this divalent cation the subunits reaggregated to the active enzyme. The different distribution patterns obtained in absence or presence of Ca showed that reactivation is directly linked to the formation of a high MW form of the protein. Substitution of Ca by other divalent cations also led to reaggregation. These aggregates are, however, inactive. The enzyme was found to be formed intracellularly in its low MW form. Experiments with protoplasts revealed that these are capable to produce and release the amylase. When the production of the enzyme by protoplasts declined, full restoration could be achieved by a recovery treatment. Normally, the enzyme released by the protoplasts consisted of equal portions of the high, medium, and low MW form of the amylase. If the cells were, however, depleted as a result of continued incubations, the extruded enzyme consisted increasingly of the low MW form, which could finally represent more than 80%. This trend could be completely reversed by the supply of carbon and nitrogen sources during the recovery treatment, whereafter the enzyme consisted again of the intially observed equal amounts of the three MW forms. Vesicles prepared from the protoplasts were also found to release amylase, but on a lower level, and only for a very limited time, with no possibility to regain activity by a recovery treatment. Subunit formation was also observed during column chromatography, which could be counteracted by a sufficient supply with Ca-ions.     

Differentiation of adreno-chromaffin cells in the newborn rat, as detected by formaldehyde-induced fluorescence, compared with the chromaffin reaction

The newborn of 4 pregnant female rats were collected within 24 h of birth. Twelve neonates were used for the study of chromaffin reaction, by both the direct and the indirect methods. The medullary cells could be demonstrated at this stage; however, the demarcation between noradrenaline and adrenaline cells was not quite sharp. Application of formaldehyde-induced fluorescence to the newborn rat adrenal gland was highly significant. A clear distinction could be demonstrated between noradrenaline and adrenaline cells, the former giving off strong, white-green fluorescence and the latter being weakly green-fluorescent. A third type giving off moderate green fluorescence was interposed between the cells and possibly represented an equivocal cell type at this stage. Sympathetic nerves could be demonstrated in relation to blood vessels and medullary cells.     

Enhanced catalysis of l-asparaginase from Bacillus licheniformis by a rational redesign

l-Asparaginase (3.5.1.1) being antineoplastic in nature are used in the treatment of acute lymphoblastic leukemia (ALL). However glutaminase activity is the cause of various side effects when used as a drug against acute lymphoblastic leukemia (ALL). Therefore, there is a need of a novel l-asparaginase (L-ASNase) with low or no glutaminase activity. Such a property has been observed with L-ASNase from B. licheniformis (BliA). The enzyme being glutaminase free in nature paved the way for its improvement to achieve properties similar to or near to the commercially available L-ASNases. Rational enzyme engineering approach resulted in four mutants: G238N, E232A, D103V and Q112H. Among these the mutant enzyme, D103V, had a specific activity of 597.7 IU/mg, which is higher than native (rBliA) (407.65 IU/mg). Moreover, when the optimum temperature and in vitro half life were studied and compared with native BliA, D103V mutant BliA was better, showing tolerance to higher temperatures and a 3 fold higher half life. Kinetic studies revealed that the mutant D103V L-ASNase has increased substrate affinity, with K_m value of 0.42 mM and V_max of 2778.9 μmol min⁻¹.     

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product.

By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolytically active phages on plates supplemented with 0.8% skim milk, chromosomal B. caldolyticus DNA fragments that specified proteolytic activity were obtained. Subcloning of one of these fragments in a protease-deficient Bacillus subtilis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb HinfI-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids. This ORF was denoted as the B. caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene. Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B. caldolyticus enzyme was a neutral protease. The B. sterothermophilus and B. caldolyticus enzymes differed at only three amino acid positions. Nevertheless, the thermostability and optimum temperature of the B. caldolyticus enzyme were 7 to 8 degrees C higher than those of the B. stearothermophilus enzyme. In a three-dimensional model of the B. stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions. The role of these residues in thermostability was analyzed by using site-directed mutagenesis. It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B. stearothermophilus and B. caldolyticus.     

Effect of partial pressure of CO2 on the production of thermostable alpha-amylase and neutral protease by Bacillus caldolyticus

Controlling the concentration of dissolved oxygen is a standard feature in aerobic fermentation processes but the measurement of dissolved CO2 concentrations is often neglected in spite of its influence on the cellular metabolism. In this work room air and room air supplemented with 5% and 10% carbon dioxide were used for aeration during the cultivation of the thermophilic microorganism Bacillus caldolyticus (DSM 405) on starch to produce alpha-amylase (E.C. 3.2.1.1) and neutral protease (E.C. 3.4.24.27/28). The increased CO2 concentrations resulted in a 22% raise in activity of secreted alpha-amylase and a 43% raise in protease activity when compared with aeration with un-supplemented room air. There was no effect on the final biomass concentration. Furthermore, the lag-phase of fermentation was reduced by 30%, further increasing the productivity of alpha-amylase production. Determinations of dissolved CO2 in the culture broth were conducted both in situ with a probe as well as using exhaust gas analysis and both the methods of quantification showed good qualitative congruence.