Purification and reversible subunit dissociation of overproduced Escherichia coli phenylalanyl-tRNA synthetase

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) has been purified to homogeneity from a 100-fold overproducing Escherichia coli strain carrying a hybrid pBR322 plasmid containing the pheS-pheT locus. The purified enzyme is identical to the phenylalanyl-tRNA synthetase isolated from an haploid strain. The enzyme was found to dissociate in the presence of 0.5 M NaSCN and the α- and β-subunits composing the native α₂β₂ enzyme were separated by gel filtration. Neither isolated subunit showed significant catalytic activity. A complex indistinguishable from the native enzyme with full catalytic activity is recovered upon mixing the subunits. The N- and C-terminal sequences and the amino acid composition of each subunit were determined. They are compared to the available data concerning the primary structure of the subunits, as deduced from nucleotide sequencing of the pheS-pheT operon.     

Subunits of RNA polymerase in function and structure. IV. Enhancing role of sigma in the subunit assembly of Escherichia coli RNA polymerase

The in vitro reconstitution of DNA-dependent RNA polymerase of Escherichia coli is markedly enhanced by the σ subunit. This conclusion is based on the following observations: (1) the core activity was higher for the enzyme reconstituted from mixtures of α, β,β′ and σ subunits than from those devoid of the σ subunit; (2) the reconstituted enzyme lacking the σ subunit could never regain full activity even when the σ subunit was supplied before assay and (3) the recovery of enzyme activity increased in proportion to the amount of σ subunit present during reconstitution.This influence of the σ subunit was also observed when reconstitution was carried out by mixing the α₂β complex and the β′ subunit, the second step in the sequence of enzyme formation. The σ subunit-dependent assembly between the α₂β complex and the β′ subunit required an ionic strength of around 0.2 m-KC1 and was enhanced by higher temperatures. In contrast, formation of the α₂β complex, which exhibited no requirement for the σ subunit, was unaffected by the salt concentration used or the temperature of reaction. The enhancement was observed not only at neutral but also at alkaline pH. The native enzyme per se was greatly activated after brief exposure to alkali.     

Thermostable succinyl-Coenzyme A synthetase from Nitrosomonas europaea ATCC 25978: Purification and properties

The ammonia-oxidizing chemoautotrophic bacterium Nitrosomonas europaea possesses prominant succinate-reducing activity of succinyl-Coenzyme A synthetase (SCS, EC 6.2.1.5). SCS was purified as an electrophoretically homogeneous protein from Nitrosomonas europaea strain ATCC 25978 about 275-fold, with a 3.9% activity yield. The molecular mass of the native enzyme was estimated to be about 130 kDa by gel filtration, whereas SDS-PAGE gave two protein bands with M_r values of 29 (α) and 36 kDa (β). The isoelectric point of the enzyme was 5.3. The apparent K_m values of the enzyme for ATP, succinate and CoA were 0.4 mM, 5 mM and 0.1 mM, respectively. The pH and temperature optima of the SCS were about 5.0 and 55°C, respectively. The SCS was stable in the pH range of 8.0–10.0 and up to 70°C. The enzyme was thermostable; 50% of the enzyme activity was retained at 90–100°C for 10 min. The SCS was activated by Mg²⁺ at 1.0–100 mM, but inhibited by Cu²⁺ (0.1 mM) and SDS (1.0 mM). The enzyme utilized ATP as the preferred substrate.     

Studies on the subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

Both uncomplexed subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium have an absolute requirement for divalent metal ions which can be satisfied by Mg²⁺, Mn²⁺, or Co²⁺. The metal ion kinetics for uncomplexed anthranilate synthetase give biphasic double-reciprocal plots and higher apparent K_m values than those for anthranilate synthetase in the enzyme complex. In contrast, the apparent K_m values for phosphoribosyltransferase are the same whether the enzyme is uncomplexed or complexed with anthranilate synthetase. This suggests that the metal ion sites on anthranilate synthetase, but not those on phosphoribosyltransferase, are altered upon formation of the enzyme complex. These results and the results of studies reported by others, suggest that complex formation between anthranilate synthetase and phosphoribosyltransferase leads to marked alterations at the active site of the former, but not the latter enzyme. Uncomplexed anthranilate synthetase can be stoichiometrically labeled with Co(III) under conditions which lead to inactivation of 75% of its activity. A comparison of the effects of anthranilate and tryptophan on phosphoribosyltransferase activity in the uncomplexed and complexed forms shows that anthranilate, but not tryptophan, inhibits the uncomplexed enzyme. The complexed phosphoribosyltransferase shows substrate inhibition by anthranilate binding to the phosphoribosyltransferase subunits. In contrast, in a tryptophan-hypersensitive variant complex, anthranilate inhibits phosphoribosyltransferase activity by acting on the anthranilate synthetase subunits. The data are interpreted to mean that there are two classes of binding sites for anthranilate, one on each type of subunit, which may participate in the regulation of anthranilate synthetase and phosphoribosyltransferase under different conditions.     

Evidence that the essential, photosensitive histidyl residue in the beta2 subunit of tryptophan synthetase is in the pyridoxyl peptide

The β₂ subunit of tryptophan synthetase of Escherichia coli is photoinactivated in the presence of pyridoxal 5′-phosphate and L-serine as a result of the destruction of one histidyl residue per chain (1). Two tryptic peptides are found in much lower amounts in the photoinactivated enzyme than in the control enzyme. These peptides have been identified from their amino acid composition as the 9 or 10 residue peptides which terminate with the lysyl residue which forms a Schiff base with pyridoxal 5′-phosphate. These peptides contain two histidyl residues, one of which appears to be photosensitive. Thus pyridoxal 5′-phosphate sensitizes the photooxidation of a nearby, essential histidyl residue.     

Antico-operative binding of bacterial and mammalian initiator tRNAMet to methionyl-tRNA synthetase from Escherichia coli

The reaction scheme of methionyl-tRNA synthetase from Escherichia coli with the initiator tRNA_s^Met from E. coli and rabbit liver, respectively, has been resolved. The statistical rate constants for the formation, k_R, and for the dissociation, k_D, of the 1:1 complex of these tRNAs with the dimeric enzyme have been calculated. Identical k_R values of 250 μm^?1 s^?1 reflect similar behaviour for antico-operative binding of both tRNA_s^Met to native methionyl-tRNA synthetase. Advantage was taken of the difference in extent of tryptophan fluorescence-quenching induced by the bacterial and mammalian initiator tRNA_s^Met to measure the mode of exchange of these tRNAs antico-operatively bound to the enzyme. Analysis of the results reveals that antico-operativity does not arise from structural asymmetric assembly of the enzyme subunits. Indeed, both subunits can potentially bind a tRNA molecule. Exchange between tRNA molecules can occur via a transient complex in which both sites are occupied. Either strong and weak sites reciprocate between subunits on the transient complex or occupation of the weak site induces symmetry of this complex. While in the present case, these two alternatives are kinetically indistinguishable, they do account for the observation that, upon increasing the concentration of the competing mammalian tRNA, the rate of exchange of the E. coli initiator tRNA^Met is enhanced, due to its faster rate of dissociation from the transient complex. Finally, it has been verified that in the case of the trypsin-modified methionyl-tRNA synthetase which cannot provide more than one binding site for tRNA, exchange of enzymebound bacterial tRNA by mammalian tRNA does proceed to a limiting rate independent of the mammalian tRNA concentration present in the solution.     

Magnetic resonance studies of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

The binding of Mn²⁺ to the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium was examined by electron paramagnetic resonance studies. Two types of binding sites were observed: one to two tight sites with a dissociation constant of 3–5 μm and five to six weaker sites with a dissociation constant of 40–70 μm. The activator constant for Mn²⁺ was found to be 9 μm for the glutamine-linked anthranilate synthetase activity and 4 μm for the phosphoribosyltransferase activity. These values are both in the range of the dissociation constant for the tight sites. Water proton relaxation rate measurements showed that the binary enhancement values for both classes of sites were equivalent, ?_b = 10.7 ± 2.0. The addition of chorismate to the Mn²⁺-enzyme complexes when predominantly the tight Mn²⁺ sites were occupied resulted in a large decrease in the observed enhancement (?_T = 2.0). Addition of 5-phosphoribosyl-1-pyrophosphate to the enzyme-Mn²⁺ complexes caused large decreases in the water proton relaxation rate (?_T = 1.5) when tight or tight plus weaker Mn²⁺ sites were occupied. No changes in the water proton relaxation rate were observed when glutamine, pyruvate, or anthranilate were added; a small decrease was observed when enzyme-Mn²⁺ was titrated with tryptophan. Tryptophan significantly altered the effect of the binding of chorismate but not of 5-phosphoribosyl-1-pyrophosphate. The effect of tryptophan on the water proton relaxation rate of a Mn²⁺-enzyme-chorismate complex using a variant enzyme complex which is tryptophan hypersensitive (P. D. Robison, and H. R. Levy, 1976, Biochim. Biophys. Acta. 445, 475–485) occurred at lower concentrations than for the normal enzyme complex. The uncomplexed anthranilate synthetase subunit was titrated with Mn²⁺ and found to have one to two binding sites with a dissociation constant of 300 ± 100 μm. This dissociation constant is much larger than the activator constant for Mn²⁺ for uncomplexed anthranilate synthetase which was determined to be 4 μm. These results indicate that the Mn²⁺-binding sites on anthranilate synthetase are altered when the enzyme complex is formed and that both chorismate and 5-phosphoribosyl-1-pyrophosphate interact closely with enzyme-bound Mn²⁺ or cause a large effect upon its environment.     

Subunits of RNA polymerase in function and structure; Maturation in vitro of core enzyme from Escherichia coli

Reconstitution of Escherichia coli RNA polymerase was found to be markedly enhanced by DNA as well as by the σ subunit. Among discrete steps of subunit assembly, formation of the primary intermediate α₂β complex and subsequent association of the complex with the β′ subunit are not affected by the presence of DNA and the σ subunit; the α₂ββ′ complex thus formed, however, is virtually inactive and is subject to temperature-dependent activation by DNA and the σ subunit. The α₂ββ′ complex is, therefore, a secondary intermediate in the sequence of enzyme formation, or a premature form of core enzyme.In the course of activation of the premature core complex, the subunit σ interacts with both the α₂β complex and the β′ subunit; DNA acts in much the same way. The enzyme, reconstituted in the presence of DNA, is recovered attached to the DNA, added as an enhancer, and initiates RNA synthesis without prior release from the DNA. A limited number of unique DNA sites appear to be concerned with the enzyme maturation.     

Carbohydrate binding specificities and crystal structure of the cholera toxin-like B-subunit from Citrobacter freundii

Enterotoxigenic Escherichia coli and Vibrio cholerae are well known causative agents of severe diarrheal diseases. Both pathogens produce AB₅ toxins, with one enzymatically active A-subunit and a pentamer of receptor-binding B-subunits. The primary receptor for both B-subunits is the GM1 ganglioside (Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer), but the B-subunits from porcine isolates of E. coli also bind neolacto-(Galβ4GlcNAcβ-)terminated glycoconjugates and the B-subunits from human isolates of E. coli (hLTB) have affinity for blood group A type 2-(GalNAcα3(Fucα2)Galβ4GlcNAcβ-)terminated glycoconjugates.     

Comparative analysis of the catalytic components in the archaeal dye-linked l-proline dehydrogenase complexes

Two types of hetero-oligomeric dye-linked l-proline dehydrogenases (α₄β₄ and αβγδ types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the β subunit (PDHβ) is responsible for catalyzing l-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHβs from α₄β₄- and αβγδ-type enzyme complexes, eight PDHβs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The α₄β₄-type PDHβs showed greater thermostability than most of the αβγδ-type PDHβs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the α₄β₄-type PDHβs utilized ferricyanide as the most preferable electron acceptor, whereas αβγδ-type PDHβs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHβs likely reflect whether they were from an αβγδ- or α₄β₄-type complex, though the wider divergence observed within αβγδ-type PDHβs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHβs did not reflect the complex type. Interestingly, the k _cat value for free α₄β₄-type PDHβ from P. horikoshii was much larger than the value for the same subunit within the α₄β₄-complex. This indicates that the isolated PDHβ could be a useful element for an electrochemical system for detection of l-proline.     

Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans

The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α₂β₂γ₂) structure with a native molecular mass of 210 kDa. It requires coenzyme B₁₂ for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K _m values for coenzyme B₁₂, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B₁₂-dependent diol dehydratase and not a glycerol dehydratase.     

Purification and characterization of a protease-resistant cellulase from Aspergillus niger

An endo-β-1,4-glucanase (EC 3.2.1.4) was purified from a culture filtrate of Aspergillus niger IFO31125 by column chromatography through TSK-gel DEAE-3SW and TSK-gel DEAE-5PW, and by gel filtration through TSK-gel G2000SW by high performance liquid chromatography. The enzyme was estimated to have a molecular weight of about 40 kDa by both gel filtration and SDS-polyacrylamide gel electrophoresis, and appeared to consist of a monomeric protein. It contained 8.9% carbohydrate. The optimal pH for activity was 6.0–7.0, and the stable pH range was 5.0–10.0. The optimum temperature at pH 6.0 was around 70°C. The enzyme was very thermally stable and no loss of original activity was found on incubation at 60°C for 2 h. The enzyme efficiently hydrolyzed carboxymethylcellulose and lichenan, but crystalline forms of cellulose, curdlan, laminarin, cellobiose, p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside were barely hydrolyzed. The activity of the enzyme was inhibited by Hg²⁺ and Cu²⁺ but was not affected by other inhibitors of thiol enzymes such as p-chloromercuribenzoate and N-ethylmaleimide. N-Bromosuccinimide showed a strong inhibitory effect, suggesting that a tryptophan residue is essential for the activity of the enzyme. The N-terminal amino acid sequence of the enzyme showed considerable homology to those of endo-β-1,4-glucanases from some other microorganisms, including Sclerotinia sclerotiorum and Schizophyllum commune. The enzyme had very strong protease-resistance, and showed no loss of activity when incubated with proteases such as Savinase at 40°C, even for 2 weeks.     

Inhibition of Escherichia coli glutamine synthetase by phosphinothricin

The inhibition of Escherichia coli glutamine synthetase by phosphinothricin [2-amino-4-(methylphosphinyl)butanoic acid] has been studied. This amino acid was observed to function as an active site directed inhibitor exhibiting time-dependent inhibition of glutamine synthetase in the presence of ATP or adenylylimidodiphosphate (AMPPNP) but not adenylyl(β,γ-methylene) diphosphonate (AMPPCP). The inactivation was observed to be pseudo-first order. Phosphinothricin was also found to inhibit the enzyme reversibly under initial rate conditions and was competitive with respect to glutamate with K_1S = 18 ± 3 μm. The inactive enzyme inhibitor complex was found to contain approximately 11 molecules of ADP and of ³²P per dodecamer using [γ-³²P]ATP. Reactivation of the inactive enzyme complex was achieved by incubating the enzyme complex in 50 mm acetate (pH 4.4), 1 m KCl, and 0.40 m (NH₄)₂SO₄. ADP, phosphinothricin, and P_i were released upon reactivation.     

Purification,catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli

3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg²⁺ or Mn²⁺, with Mn²⁺ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40°C the K_m of E. coli IPMDH was 105 μM for IPM and 321 μM for NAD, the k_cat was 69 s⁻¹. The half denaturationn temperature was 64°C, which was 20°C lower than that of the thermophile enzyme.     

Characterization flavanone 3β-hydroxylase expressed from <Emphasis Type="Italic">Populus euphratica</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in dihydroflavonol production

Flavanone 3β-hydroxylase plays very important role in the biosynthesis of flavonoids. A putative flavanone 3β-hydroxylase gene (Pef3h) from Populus euphratica was cloned and over-expressed in Escherichia coli. Induction performed with 0.1 mM IPTG at 20°C led to localization of PeF3H in the soluble fraction. Recombinant enzyme was purified by Ni-NTA affinity. The optimal activity of PeF3H was revealed at pH 7.6 and 35°C. The purified enzyme was stable over pH range of 7.6–8.8 and had a half-life of 1 h at 50°C. The activity of PeF3H was significantly enhanced in the presence of Fe²⁺ and Fe³⁺. The K _M and V _max for the enzyme using naringenin as substrate were 0.23 mM and 0.069 μmoles mg–1min-1, respectively. The K _m and V _max for eriodictyol were 0.18 mM and 0.013 μmoles mg^–1min^–1, respectively. The optimal conditions for naringenin bioconversion in dihydrokaempferol were obtained: OD₆₀₀ of 3.5 for cell concentration, 0.1 mM IPTG, 5 mM α-ketoglutaric acid and 20°C. Under the optimal conditions, naringenin (0.2 g/L) was transformed into 0.18 g/L dihydrokaempferol within 24 h by the recombinant E. coli with a corresponding molar conversion of 88%. Thus, this study provides a promising flavanone 3β-hydroxylase that may be used in biosynthetic applications.     

Subunit structure of anthranilate synthase from Neurospora crassa: Preparation and characterization of a protease-free form

The multifunctional enzyme complex anthranilate synthase from Neurospora crassa has been purified to homogeneity by a new procedure which yields a stable preparation of the enzyme. Unlike earlier preparations of the enzyme, anthranilate synthase prepared by this technique is not degraded during incubation at 37 °C or during freeze-thaw treatment. Purified anthranilate synthase contains two subunits of M_r 84,000 (β-subunit) and 76,000 (α-subunit), which are shown, by partial proteolysis, to be unrelated in sequence. Immunoprecipitation studies demonstrate that freshly prepared crude extracts of Neurospora contain anthranilate synthase subunits identical in size with those of the purified enzyme. The β-subunit is shown to be the product of the trp1 gene, and the a-subunit, of the trp2 gene.     

Expression of a keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli and characterization of the recombinant enzymes

The gene kerA (1,047 bp) encoding the main keratinase from Bacillus licheniformis was cloned into two conventional vectors, pET30α and pET32α, and expressed in Escherichia coli. From SDS-PAGE analysis, the recombinant keratinases were 45 and 55 kDa. They had different optimal pH values (7.5 and 8.5) but the same optimum temperature of 50 °C. The recombinant keratinase produced in E. coli pET30α-kerA was more stable than that produced in E. coli pET32α-kerA, and retained approx. 70 % of its total enzyme activity after 30 min at 70 °C.     

Structural and biochemical properties of lichenase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu²⁺ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu²⁺ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni²⁺, Co²⁺ and Zn²⁺ did not affect the activity of the enzyme at low concentrations (0–10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (β/α)₈-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.     

Co-clustering and internalization of low-density lipoproteins and alpha 2-macroglobulin in human skin fibroblasts

The endocytosis of low density lipoprotein (LDL) and α₂-macroglobulin (α₂M) has been examined simultaneously in human skin fibroblasts. Incubation of cells at 4 °C with rhodamine-α₂M and LDL plus [(dichlorotriazinyl)amino]fluorescein-anti-LDL gave a weak fluorescence for α₂M and a brighter, clustered fluorescence for LDL. Following warming to 37 °C, LDL and α₂M were observed to be coincident within endocytotic vesicles in the cell. By electron microscopy, LDL-ferritin and α₂M-colloidal gold were present in the same coated pit at 4 °C. After 7 min at 37 °C, both ligands were observed in the same receptosome. Pretreatment of fibroblasts at 37 °C with 200–300 μM dansylcadaverine or 50 mM methylamine blocked clustering and internalization of both LDL and α₂M. Bacitracin (5 mg ml^?) blocked clustering and endocytosis of α₂M, but not of LDL. These data indicate that both LDL and α₂M are processed via the same endocytotic pathway in skin fibroblasts.