Purification and characterization of a new L-methioninase from solid cultures of <Emphasis Type="Italic">Aspergillus flavipes</Emphasis>

L-Methioninase was purified to electrophoretic homogeneity from cultures of Aspergillus flavipes using anion-exchange and gel filtration chromatography by 12.1 fold compared to the crude enzyme preparation. The purified enzyme had a molecular mass of 47 kDa under denaturing conditions and an isoelectric point of 5.8 with no structural glycosyl residues. The enzyme had optimum activity at pH 7.8 and pH stability from 6.8–8.0 at 35°C. The enzyme appeared to be catalytically stable below 40°C. The enzyme activity was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, 2-mercaptoethanol, Hg⁺, Cu²⁺, and Fe²⁺, with slight inhibition by Triton X-₁₀₀. A flavipes L-methioninase has a higher catalytic affinity towards L-methionine (Km, 6.5 mM and Kcat, 14.1 S⁻¹) followed by a relative demethiolating activity to L-homo-cysteine (Km, 12 mM and Kcat, 9.3 S⁻¹). The enzyme has two absorption maxima at 280 and 420 nm, typical of other PLP-enzymes. Apo-L-methioninase has the ability to reconstitute its structural catalytic state completely upon addition of 0.15 mM PLP. L-Methioninase has neither an appreciable effect on liver function, platelet aggregation, nor hemolysis of human blood. The purified L-methioninase from solid cultures of A. flavipes displayed unique biochemical and catalytic properties over the currently applied Pseudomonad enzyme.     

Imidazolium chloride‐based ionic liquid‐assisted improvement of lipase activity in organic solvents

The activity of a lipase from a newly isolated Pseudomonas sp. was investigated in the presence of organic solvents and imidazolium chloride‐based ionic liquids (IL) such as BMIM[Cl] and HMIM[Cl]. The lipase activity in the presence of IL was higher compared to that in common organic solvents such as methanol and 2‐propanol. A possible explanation for the enzyme activation might be the structural changes induced in the protein in organic systems. Since IL quench the intensity of fluorescence emission, it was not possible to investigate the major factor that influences the enzyme behavior in these new organic salts. Furthermore, the enzyme exhibited excellent activity in buffer mixtures containing both organic solvent and IL. The stability of the lipase at 50°C was considerably increased in the presence of 20% BMIM[Cl] compared with the untreated lipase in aqueous medium. The light scattering method clearly showed that prevention of aggregation could be the reason for thermal stabilization at 50°C in reactions containing IL. Kinetic analysis of the enzyme in the presence of different concentrations of IL showed that the K_m value increased from 0.45 mM in aqueous buffer to 2.4 mM in 50% v/v BMIM[Cl]/buffer. The increase in K_m indicates that IL can significantly reduce the binding affinity of the substrate to the enzyme. Also, a linear correlation was observed between the BMIM[Cl] concentration and V_max of the enzyme. As the concentration of BMIM[Cl] increased from 10 to 50% v/v, the V_max value increased from 1.8 to 46 μM/min.     

Biophysical characterization of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> phosphoserine aminotransferase (EhPSAT): role of cofactor and domains in stability and subunit assembly

We investigated the role of the cofactor PLP and its binding domain in stability and subunit assembly of phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica. Presence of cofactor influences the tertiary structure of EhPSAT because of the significant differences in the tryptophan microenvironment and proteolytic pattern of holo- and apo-enzyme. However, the cofactor does not influence the secondary structure of the enzyme. Stability of the protein is significantly affected by the cofactor as holo-enzyme shows higher T _m and C _m values for thermal and GdnHCl-induced denaturation, respectively, when compared to the apo-enzyme. The cofactor also influences the unfolding pathway of the enzyme. Although urea-dependent unfolding of both holo- and apo-EhPSAT is a three-state process, the intermediates stabilized during unfolding are significantly different. For holo-EhPSAT a dimeric holo-intermediate was stabilized, whereas for apo-EhPSAT, a monomeric intermediate was stabilized. This is the first report on stabilization of a holo-dimeric intermediate for any aminotransferase. The isolated PLP-binding domain is stabilized as a monomer, thus suggesting that either the N-terminal tail or the C-terminal domain of EhPSAT is required for stabilization of dimeric configuration of the wild-type enzyme. To the best of our knowledge, this is a first report investigating the role of PLP and various protein domains in structural and functional organization of a member of subgroup IV of the aminotransferases.     

Equilibrium unfolding of <Emphasis Type="Italic">A. niger</Emphasis> RNase: pH dependence of chemical and thermal denaturation

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A_MG1) into another (I_MG2).     

An Irreversible and Kinetically Controlled Process: Thermal Induced Denaturation of <Emphasis Type="SmallCaps">L</Emphasis>-2-Hydroxyisocaproate Dehydrogenase from <Emphasis Type="Italic">Lactobacillus confusus</Emphasis>

The thermal denaturation of Lactobacillus confusus l-2-Hydroxyisocaproate Dehydrogenase (l-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and the T _m is dependent on the scan-rate, which suggests that the denaturation process of l-HicDH is kinetically determined. The heat capacity function of l-HicDH shows a single peak with the T _m values between 52.14°C and 55.89°C at pH 7.0 at different scan rates. These results indicate that the whole l-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation pathway was described by a three-state model, N → U → F, in which the dissociation of the tetramer takes place as an irreversible step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure. Thermal unfolding of l-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values.     

Biophysical studies of an NAD(P)<Superscript>+</Superscript>-dependent aldehyde dehydrogenase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Aldehyde dehydrogenase (ALDH) catalyzes the conversion of aldehydes to the corresponding acids by means of an NAD(P)⁺-dependent virtually irreversible reaction. In this investigation, the biophysical properties of a recombinant Bacillus licheniformis ALDH (BlALDH) were characterized in detail by analytical ultracentrifuge (AUC) and various spectroscopic techniques. The oligomeric state of BlALDH in solution was determined to be tetrameric by AUC. Far-UV circular dichroism analysis revealed that the secondary structures of BlALDH were not altered in the presence of acetone and ethanol, whereas SDS had a detrimental effect on the folding of the enzyme. Thermal unfolding of this enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ~0.2 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]_{05, N-U}, at 0.93 M. BlALDH was active at concentrations of urea below 2 M, but it experienced an irreversible unfolding under 8 M denaturant. Taken together, this study provides a foundation for the future structural investigation of BlALDH, a typical member of ALDH superfamily enzymes.     

Purification and partial characterization of thermostable serine alkaline protease from a newly isolated<Emphasis Type="Italic">Bacillus subtilis</Emphasis> PE-11

The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolatedBacillus subtilis PE-11. The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 gel permeation chromatography. The enzyme was shown to have a relative low molecular weight of 15 kd by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and was purified 21-fold with a yield of 7.5%. It was most active at 60°C, pH 10, with casein as substrate. It was stable between pH 8 and 10. This enzyme was almost 100% stable at 60°C even after 350 minutes of incubation. It was strongly activated by metal ions such as Ca²⁺, Mg⁺², and Mn⁺². Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP) but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with iodoacetate,p-chloromercuric benzoate (pCMB), and β-mercaptoethanol (β-ME). The compatibility of the enzyme was studied with commercial and local detergents in the presence of 10mM CaCl₂ and 1M glycine. The addition of 10mM CaCl₂ and 1M glycine, individually and in combination, was found to be very effective in improving the enzyme stability where it retained 52% activity even after 3 hours. This enzyme improved the cleansing power of various detergents. It removed blood stains completely when used with detergents in the presence of 10mM CaCl₂ and 1M glycine.     

Heat-stable pullulanase from Bacillus acidopullulyticus: characterization and refolding after guanidinium chloride-induced unfolding

Heat-stable pullulanase from Bacillus acidopullulyticus was characterized with respect to its stability against thermal and chemical denaturation and its reactivation after complete chemical unfolding. The enzyme was quite thermostable and retained 55% of activity after heating at 60°C for 30 min at pH 5.5. At pH 6.0, only 9% residual activity was observed. The addition of sucrose, polyols, and Na₂SO₄ strongly stabilized the enzyme against thermal inactivation. The processes of chemical unfolding by guanidinium chloride (GdmCl) and refolding were studied by enzymological and spectroscopic criteria. B. acidopullulyticus pullulanase was very sensitive to GdmCl denaturation and had a transition midpoint at 1.2 M GdmCl. Reactivation after complete unfolding in 5 M GdmCl was initiated by dilution of the unfolding mixture; 67% reactivation was observed under standard conditions. The influence of some chemical and physical parameters (pH, chemical agents, temperature, and unfolding and refolding time) on refolding was investigated. Of the additives tested to assist reactivation, only bovine serum albumin (BSA) increased the yield of activity to 80%. The full regain of structure and activity was proven by comparing the enzymological, physicochemical, and spectroscopic properties of the native and refolded pullulanase. Received: June 22, 1998 / Accepted: December 11, 1998     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K _m, V _max, and K _cat of the enzyme were 4.727 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.     

Purification and characterization of a mesophilic organic solvent tolerant lipase produced by Acinetobacter sp. K5b4

The extracellular lipase produced by Acinetobacter sp. K5b4 was purified to homogeneity using ultrafiltration (cutoff 30?KDa) followed by gel filtration chromatography on Sephadex G-50. The enzyme was purified to homogeneity with an apparent molecular mass of 133?KDa by SDS-PAGE. This purification resulted on 10.24 fold with 18.3% recovery. The K_m and V_max of purified enzyme when using pNPL hydrolysis were 4.0?mM and 73.53?nmol/ml/min, respectively. The pure enzyme was greatly stimulated in the presence of 20, 40 and 60% (v/v) methanol, DMSO and acetone whereas, ethanol, acetonitrile and propanol decreased the enzyme activity. Maximum enzyme activity was achieved at pH 7.0 and incubation temperature of 27?°C. The enzyme was stable within a pH range of 6.5 to 7 at 27?°C for 1?h. The enzyme activity was enhanced up to 36% by KCl, BaCl₂, MgCl₂ and CaCl₂ while obviously inhibited (10–20%) by CoCl₂, ZnCl₂, MnCl₂ and CuCl₂. No inhibitory effects were observed with 1.0 and 5.0?mM of 2-mercaptoethanol and EDTA. Similarly, SDS at 1.0?mM does not affect the enzyme activity while high reduction (80%) was observed at 5.0?mM SDS concentration. The enzyme was active against p-nitrophenyl esters of C8, C12 and C16 with highest preference to the medium carbon chain p-nitrophenyl caprylate (C8). The fact that the enzyme displays distinct stability in the presence of methanol, DMSO and acetone suggests that this lipase is suitable as biocatalyst in organic synthesis where such hydrophilic organic solvents are used as a reaction media.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (T_m ≈ 45 °C) than does the kidney enzyme (T_m ≈ 55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Thermostable lipoxygenase is a key enzyme in the conversion of linoleic acid to trihydroxy-octadecenoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Lipoxygenases (LOXs) constitute a family of lipid-peroxidizing enzymes that catalyze the oxidation of unsaturated fatty acid containing a (1Z,4Z)-pentadiene structural unit, leading to formation of conjugated (Z,E)-hydroperoxydienoic acid. LOXs are known to be widely distributed in plants and animals. Recently, several microbial LOXs were reported to be involved in the production of hydroperoxy fatty acids. Among the microorganisms that produce hydroxy fatty acids, Pseudomonas aeruginosa PR3 is known to convert linoleic acid to trihydroxy fatty acid, which suggests the involvement of a LOX enzyme. Based on these reports, we identified a novel thermostable LOX from P. aeruginosa PR3 strain. The protein was purified 34.3-fold with a recovery rate of 5.14%. The K_m and V_max values of the purified enzyme were 3.57 mM and 0.73 μmol/min//mg, respectively. Heat stability of the purified enzyme was unexpectedly high with an LD₅₀ of 90 min at 80°C, although P. aeruginosa PR3 is known as a mesophilic bacterium. Substrate specificity of the purified enzyme was restricted only to unsaturated fatty acids carrying a (1Z,4Z)-pentadiene unit.     

Thermal stability and biochemical properties of isocitrate dehydrogenase from the thermoacidophilic archaeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Isocitrate dehydrogenase [IDH; EC 1.1.1.42] from the thermoacidophilic archaeon Thermoplasma acidophilum (TaIDH) showed high thermal stability with an apparent melting temperature, T _m, of 82.2 and 84.5°C at pH 7.5 and 5.8, respectively. Based on structural alignment of TaIDH with IDH from Aeropyrum pernix (ApIDH) and Archaeoglobus fulgidus (AfIDH) residues forming an aromatic cluster in the clasp-domain thought to strengthen the dimer interface in ApIDH and AfIDH were identified in the former enzyme. Moreover, TaIDH had a shortened N-terminus that may protect the enzyme from thermal denaturation. The enzyme activity of TaIDH was highest at 70°C. The pH-activity profile was bell-shaped with an optimum shifted to a lower pH compared to AfIDH. The activity of TaIDH was influenced by changes in pH with a three-fold reduction in activity when the pH was shifted from the pH-optimum at 7.5 to pH 5.8. However, the specific activity at pH 5.8 was still high when compared with AfIDH. The reduction in activity at pH 5.8 was not due to instability of the enzyme as the T _m of TaIDH was higher at pH 5.8 than at 7.5 and the enzyme retained 91% of its activity after incubation at 1 h at pH 5 and 60°C. The difference in the pH-profile of TaIDH in comparison with AfIDH may thus be related to the pK _as of their catalytic residues involved in the initial proton abstraction and the final proton donation during the catalysis of oxidative decarboxylation of isocitrate to 2-oxoglutarate and reduced coenzyme.     

Modification of Lys-6 and Lys-65 Affects the Structural Stability of Taiwan Cobra Phospholipase A2

To assess whether chemical modification of phospholipase A₂ (PLA₂) enzymes may affect their fine structure and consequently alter their enzymatic activity, the present study was carried out. Both Lys-6 and Lys-65 in the Taiwan cobra (Naja naja atra) PLA₂ were selectively modified with trinitrobenzene sulfonate and pyridoxal-5′-phosphate (PLP), respectively. Incorporation of either trinitrophenylated (TNP) or PLP groups on Lys-6 and Lys-65 caused a drop in PLA₂ activity, but the Ca²⁺-binding ability and global conformation of modified derivatives were not significantly different from that of native enzyme. A distinct enhancement of stability was observed with native PLA₂ when thermal unfolding was conducted in the presence of 20 mM Ca²⁺. Conformational transition induced by guanidine hydrochloride was also attenuated by the addition of Ca²⁺. Conversely, a marked decrease in the structural stability was noted with modified derivatives, and the enhancing effect of Ca²⁺ pronouncedly decreased. Together with the finding that the incorporated TNP and PLP groups did not equally affect enzymatic activity and structural stability of PLA₂, our data suggest that an alteration in the fine structure owing to the incorporated groups should contribute to the observed decrease in PLA₂ activity.     

Directed evolution of <Emphasis Type="Italic">Streptomyces lividans</Emphasis> xylanase B toward enhanced thermal and alkaline pH stability

The thermal and alkaline pH stability of Streptomyces lividans xylanase B was improved greatly by random mutagenesis using DNA shuffling. Positive clones with improved thermal stability in an alkaline buffer were screened on a solid agar plate containing RBB-xylan (blue). Three rounds of directed evolution resulted in the best mutant enzyme 3SlxB6 with a significantly improved stability. The recombinant enzyme exhibited significant thermostability at 70°C for 360 min, while the wild-type lost 50% of its activity after only 3 min. In addition, mutant enzyme 3SlxB6 shows increased stability to treatment with pH 9.0 alkaline buffer. The K _m value of 3SlxB6 was estimated to be similar to that of wild-type enzyme; however k _cat was slightly decreased, leading to a slightly reduced value of k _cat/K _m, compared with wild-type enzyme. DNA sequence analysis revealed that eight amino acid residues were changed in 3SlxB6 and substitutions included V3A, T6S, S23A, Q24P, M31L, S33P, G65A, and N93S. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Our results suggest that DNA shuffling is an effective approach for simultaneous improvement of thermal and alkaline pH stability of Streptomyces lividans xylanase B even without structural information.     

Effects of C-terminal truncation on autocatalytic processing of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> γ-glutamyl transpeptidase

The role of the C-terminal region of Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT) was investigated by deletion analysis. Seven C-terminally truncated BlGGTs lacking 581–585, 577–585, 576–585, 566–585, 558–585, 523–585, and 479–585 amino acids, respectively, were generated by site-directed mutagenesis. Deletion of the last nine amino acids had no appreciable effect on the autocatalytic processing of the enzyme, and the engineered protein was active towards the synthetic substrate L-γ-glutamyl-p-nitroanilide. However, a further deletion to Val576 impaired the autocatalytic processing. In vitro maturation experiments showed that the truncated BlGGT precursors, pro-Δ(576–585), pro-Δ(566–585), and pro-Δ(558–585), could partially precede a time-dependent autocatalytic process to generate the L- and S-subunits, and these proteins showed a dramatic decrease in catalytic activity with respect to the wild-type enzyme. The parental enzyme (BlGGT-4aa) and BlGGT were unfolded biphasically by guanidine hydrochloride (GdnCl), but Δ(577–585), Δ(576–585), Δ(566–585), Δ(558–585), Δ(523–585), and Δ(479–585) followed a monophasic unfolding process and showed a sequential reduction in the GdnCl concentration corresponding to half effect and ΔG ₀ for the unfolding. BlGGT-4aa and BlGGT sedimented at ∼4.85 S and had a heterodimeric structure of approximately 65.23 kDa in solution, and this structure was conserved in all of the truncated proteins. The frictional ratio (f/f _o) of BlGGT-4aa, BlGGT, Δ(581–585), and Δ(577–585) was 1.58, 1.57, 1.46, and 1.39, respectively, whereas the remaining enzymes existed exclusively as precursor form with a ratio of less than 1.18. Taken together, these results provide direct evidence for the functional role of the C-terminal region in the autocatalytic processing of BlGGT.     

Adenosine 5‘-Phosphosulfate Sulfotransferase from Norway Spruce: Biochemical and Physiological Properties*

Biochemical and physiological properties of adenosine 5′-phosphosulfate sulfotransferase, a key enzyme of assimilatory sulfate reduction, from spruce trees growing under field conditions were studied. The apparent K_m for adenosine 5′-phosphosulfate (APS) was 29 ± 5.5μM, its apparent M_r was 115,000. 5′-AMP inhibited the enzyme competitively with a K_i of 1 mM, but also stabilized it. MgS0₄ at 800 mM increased adenosine 5′-phosphosulfate sulfotransferase activity by a factor of 3, concentrations higher than lOOOmM were inhibitory. Treatment of isolated shoots with nutrient solution containing 1 or 2 mM sulfate, and 3 or 10 mM glutathione, respectively, induced a significant decrease in extractable adenosine 5′-phosphosulfate sulfotransferase activity over 24h, whereas GSH as well as S^2- up to 5mM cysteine and up to 200 mM SO₃^2- had no effect on the in vitro activity of the enzyme. As with other enzymes involved in assimilatory sulfate reduction, namely ATP sulfurylase (EC 2.7.7.4), sulfite reductase (EC 1.8.7.1) and O-acetyl-L.-serine sulfhydrylase (EC 4.2.99.8), adenosine 5′-phosphosulfate sulfotransferase was still detected at appreciable activities in 2- and 3-year-old needles. Adenosine 5′-phosphosulfate sulfotransferase activity was low in buds and increased during shoot development, parallel to the chlorophyll content. The enzyme activity was characterized by an annual cycle of seasonal changes with an increase during February and March.     

Purification and Characterization of Nitrate Reductase from the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

Summary Factors affecting the activity of nitrate reductase (E.C.1.7.7.2) from the halotolerant cyanobacterium Aphanothece halophytica were investigated. Cells grown in nitrate-containing medium exhibited higher nitrate reductase activity than cells grown in medium in which nitrate was replaced by glutamine. When ammonium was present in the medium instead of nitrate, the activity of nitrate reductase was virtually non-detectable, albeit with normal cell growth. The enzyme was localized mainly in the cytoplasm. The enzyme was purified 406-fold with a specific activity of 40.6 μmol/min/mg protein. SDS-PAGE revealed a subunit molecular mass of 58 kDa. Gel filtration experiments revealed a native molecular mass of 61 kDa. The K _m value for nitrate was 0.46 mM. Both methyl viologen and ferredoxin could serve as electron donor with K _m values of 4.3 mM and 5.2 μM, respectively. The enzyme was strongly inhibited by sulfhydryl-reactive agents and cyanide. Nitrite, the product of the enzyme reaction, showed little inhibition. Chlorate, the substrate analog, could moderately inhibit the enzyme activity. NaCl up to 200 mM stimulated the activity of the enzyme whereas enzyme inhibition was observed at ≥300 mM NaCl.