Streptomyces beta-alanine:alpha-ketoglutarate aminotransferase, a novel omega-amino acid transaminase. Purification, crystallization, and enzymologic properties

An enzyme which catalyzes the transamination of beta-alanine with alpha-ketoglutarate was purified to homogeneity from Streptomyces griseus IFO 3102 and crystallized. Molecular weight of the enzyme was found to be 185,000 +/- 10,000 by a gel-filtration method. The enzyme consists of four subunits identical in molecular weight (51,000 +/- 1,000). The transaminase is composed of 483 amino acids/subunit containing 7 and 8 residues of half-cystine and methionine, respectively. The enzyme exhibits absorption maxima at 278 and 415 nm. The pyridoxal 5'-phosphate content was determined to be 4 mol/mol of enzyme. The enzyme catalyzes transamination of omega-amino acids including taurine and hypotaurine. beta-Alanine and DL-beta-aminoisobutyrate served as a good amino donor; the Michaelis constants are 8.0 and 12.5 mM, respectively. alpha-Ketoglutarate is the only amino acceptor (Km = 4.0 mM); pyruvate and oxalacetate are inactive. Based on the substrate specificity, the terminology of beta-alanine:alpha-ketoglutarate transaminase is proposed for the enzyme. Carbonyl reagents, HgCl2,DL-gabaculine, and alpha-fluoro-beta-alanine strongly inhibited the enzyme.     

Growth factor stimulation of phospholipase C-gamma 1 activity. Comparative properties of control and activated enzymes.

We demonstrated previously tyrosine phosphorylation-dependent modulation of phospholipase C-gamma 1 (PLC-gamma 1) catalytic activity (Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M. T., Tonks, N. K., Rhee, S. G., and Carpenter, G. (1990) Science 250, 1253-1256). The increase in PLC-gamma 1 catalytic activity in A-431 cells occurs rapidly, with maximal activation 5 min after epidermal growth factor (EGF) stimulation. Certain other growth factors (fibroblast growth factor, platelet-derived growth factor) also stimulate PLC-gamma 1 catalytic activity, whereas insulin does not. A similar increase in PLC-gamma 1 specific activity (2-3-fold) was observed in both soluble (cytosol) and particulate (membrane) preparations from EGF-treated cells. Tyrosine-phosphorylated PLC-gamma 1 was detected in both cytosol and membrane fractions in lysates from EGF-treated A-431 cells, but the proportion of tyrosine-phosphorylated PLC-gamma 1 was higher in the cytosol (approximately 50%) than in the membrane (approximately 20%). Because a micellar concentration of the non-ionic detergent Triton X-100 allows detection of the tyrosine phosphorylation-dependent increase in PLC-gamma 1 catalytic activity in this assay, we evaluated the kinetic properties of PLC-gamma 1, immunoprecipitated from cytosol of control or EGF-treated cells, using substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2), solubilized in Triton X-100 at various molar ratios. The behavior of the control enzyme differed from the EGF-activated enzyme with respect to both Ks and Km. The control enzyme has a 7.5-fold higher Ks value than the activated enzyme (1.5 mM as compared with 0.22 mM). Activation by EGF is also a positive allosteric modifier of PLC-gamma 1-catalyzed PtdIns 4,5-P2 hydrolysis, i.e. the activated enzyme displayed apparent Michalis-Menton kinetics, with a Km of 0.6 mol fraction PtdIns 4,5-P2, whereas the control enzyme displayed sigmoidal kinetics with respect to PtdIns 4,5-P2 hydrolysis. At low substrate mol fractions (e.g. 0.07), the reaction velocity of the control enzyme was 4-fold lower than the activated enzyme. However, at a high substrate mol fraction (e.g. 0.33), the estimated maximal reaction velocities (Vmax) for both forms of PLC-gamma 1 were equivalent. PLC-gamma 1 activity from both control and EGF-treated cells was stimulated by increasing nanomolar Ca2+ concentrations. Although the catalytic activity of PLC-gamma 1 from EGF-treated cells was greater than control PLC-gamma 1 at every Ca2+ concentration tested, the relative stimulation of activity was markedly greater at Ca2+ concentrations above approximately 300 nM.     

Thermostable dipeptidase from Bacillus stearothermophilus: its purification, characterization, and comparison with aminoacylase

Dipeptidase (dipeptide hydrolase [EC 3.4.13.11]) has been purified to homogeneity and crystallized from the cell extract of Bacillus stearothermophilus IFO 12983. The enzyme has a molecular weight of about 86,000, and is composed of two subunits identical in molecular weight (43,000). The enzyme contains 2 g atoms of zinc per mol of protein. A variety of dipeptides consisting of glycine or only L-amino acids serve as substrates of the enzyme; Km and Vmax values for L-valyl-L-alanine are 0.5 mM and 68.0 units/mg protein, respectively. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturants such as urea and guanidine hydrochloride. The enzyme also catalyzes hydrolysis of several N-acylamino acids with Vmax values 3-30% of those for the hydrolysis of dipeptides. The thermostable dipeptidase shares various properties with bacterial aminoacylase [EC 3.5.1.14]: their subunit molecular weight, metal content and requirement, amino acid composition, and amino acid sequence in the N-terminal region are very similar.     

Acyl-peptide hydrolase from rat liver. Characterization of enzyme reaction

Acyl-peptide hydrolase, which catalyzes the hydrolysis of an N-terminally acetylated peptide to release an N-acetylamino acid, was isolated from rat liver and found to be N-terminally blocked. The kinetics of the hydrolysis of acetyl (Ac)-Ala-Ala, Ac-Ala-Ala-Ala, acetylalanine p-nitroanilide, and acetylalanine beta-naphthylamide were investigated. The Km values were between 1 and 9 mM, and the Vmax values were between 100 and 500 nmol/min/micrograms of enzyme. The enzyme activity toward acetylalanine p-nitroanilide and acetylalanine beta-naphthylamide was activated by the presence of Cl- and SCN- at concentrations between 0.1 and 0.5 M. By contrast, the activity toward Ac-Ala-Ala and Ac-Ala-Ala-Ala was inhibited by these anions. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by the addition of diisopropyl fluorophosphate, diethyl pyrocarbonate. Woodward's Reagent K, and glycine methyl ester/carbodiimide. Titration by diisopropyl fluorophosphate showed 0.7 mol of active serine/mol of enzyme subunit, which was confirmed by the incorporation of [3H]diisopropyl fluorophosphate into the enzyme. Acetylalanine chloromethyl ketone inactivated the enzyme following pseudo-first order kinetics; and Ac-Ala, a competitive inhibitor, protected the enzyme from this inactivation. Acyl-peptide hydrolase appears to be a serine protease utilizing a charge relay system involving serine, histidine, and, probably, a carboxyl group(s). Two series of acetyl dipeptides, acetylamino acid p-nitroanilides and acetylamino acid beta-naphthylamides, were prepared in order to determine enzyme specificity. The enzyme preferentially removed Ac-Ala, Ac-Met, and Ac-Ser, the most common acetylated N-terminal residues (Persson, B., Flinta, C., von Heijne, G., and J?rnvall, H. (1985) Eur. J. Biochem. 152, 523-527). The enzyme was shown to be useful for deblocking peptides (e.g. alpha-melanocyte-stimulating hormone and acetyl-renin substrate), and the crude enzyme/substrate mixtures were amenable to direct protein sequence analysis.     

Purification, characterization, and gene cloning of a novel fluoroacetate dehalogenase from Burkholderia sp. FA1

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of the highly stable carbon–fluorine bond in an aliphatic compound. The bacterial isolate FA1, which was identified as Burkholderia, grew on fluoroacetate as the sole carbon source to produce fluoroacetate dehalogenase (FAc-DEX FA1). The enzyme was purified to homogeneity and characterized. The molecular weights were estimated to be 79,000 and 34,000 by gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that the enzyme is a dimer. The purified enzyme was specific to haloacetates, and fluoroacetate was the best substrate. The activities toward chloroacetate and bromoacetate were less than 5% of the activity toward fluoroacetate. The K_m and V_max values for the hydrolysis of fluoroacetate were 5.1 mM and 11 μmol per minute milligram, respectively. The gene coding for the enzyme was isolated, and the nucleotide sequence was determined. The open reading frame consisted of 912 nucleotides, corresponding to 304 amino acid residues. Although FAc-DEX FA1 showed high sequence similarity to fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX H1) (61% identity), the substrate specificity of FAc-DEX FA1 was significantly different from that of FAc-DEX H1: FAc-DEX FA1 was more specific to fluoroacetate than FAc-DEX H1.     

Cloning,expression, and purification of choline dehydrogenase from the moderate halophile Halomonas elongata

Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate. Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures. To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro. In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase. The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose. The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 micro mol of O(2) min(-1) mg(-1) mM(-1) at pH 7 and 25 degrees C, respectively. With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent V(max) values for choline and betaine-aldehyde were 10.9 and 5.7 micro mol of O(2) min(-1) mg(-1), respectively. These V(max) values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor. Altogether, the kinetic data are consistent with the conclusion that H. elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available.     

Isolation, crystallization, and properties of indolyl-3-alkane alpha-hydroxylase. A novel tryptophan-metabolizing enzyme.

Indolyl-3-alkane alpha-hydroxylase, a novel tryptophan-metabolizing enzyme, was prepared in crystalline form from soil isolate organism Pseudomonas XA. Emission spectroscopy and atomic absorption analyses of purified enzyme revealed the presence of iron (0.8 mol/mol of protein), and a number of observations supported the presence of heme prosthetic group (1.1 mol/mol of protein). The S20,w value of indolyl-3-alkane alpha-hydroxylase is 10.2 S, and the molecular weight by sedimentation equilibrium ultracentrifugation is 250,000. The E1%280 of the enzyme is 21, and the isoelectric point by isoelectric focusing on ampholine polyacrylamide gel plates is 4.8. The enzyme catalyzes hydroxylation on the side chain of a variety of 3-substituted indole compounds, including certain tryptophan-containing oligopeptides. The reaction product from tryptamine was identified by proton nuclear magnetic resonance and gas chromatography/mass spectroscopy analyses. While the indole ring remained intact, hydroxylation occurred at the side chain carbon adjacent to the ring. Nuclear magnetic resonance studies indicated that hydroxylation always took place at the same position when the substrate was tryptophan methyl ester, tryptophol, indole-3-propionate, or indole-3-butyrate. No other chemical change occurred when these substrates were incubated with the enzyme. The Km value of indolyl-3-alkane alpha-hydroxylase for L-tryptophan is 2.4 X 10(-6) M, at pH 7.2. The enzyme is inhibited by potassium cyanide (0.1 mM) or hydroxylamine (1mM), but not by NaBH4 (25 mM), aminooxyacetic acid (7mM), quinacrine (1 mM), chlortetracycline (1 mM), p-mercuribenzoate (0.1 mM), or ethylenediaminetetraacetate (1 mM). The plasma half-life (t1/2) of indolyl-3-alkane alpha-hydroxylase in tumor-bearing mice is approximately 25 h.     

Properties of taurine: alpha-ketoglutarate aminotransferase of Achromobacter superficialis. Inactivation and reactivation of enzyme

The activity of taurine: alpha-ketoglutarate aminotransferase (taurine: 2-oxoglutarate aminotransferase, EC 2.6.1.55) from Achromobacter superficialis is significantly diminished by treatment of the enzyme with (NH4)2SO4 in the course of purification, and recovered by incubation with pyridoxal phosphate at high temperatures such as 60 degrees C. The inactive form of enzyme absorbing at 280 and 345 nm contains 3 mol of pyridoxal phosphate per mol. The activated enzyme contains additional 1 mol of pyridoxal phosphate with a maximum at 430 nm. This peak is shifted to about 400 nm as a shoulder by dialysis of the enzyme, but the activity is not influenced. The inactive form is regarded as a partially resolved form, i.e. a semiapoenzyme. The enzyme catalyzes transamination of various omega-amino aicds with alpha-ketoglutarate, which is the exclusive amino acceptor. Hypotaurine, DL-beta-aminoisobutyrate, beta-alanine and taurine are the preferred amino donors. The apparent Michaelis constants are as follows; taurine 12 mM, hypotaurine 16 mM, DL-beta-aminoisobutyrate 11 mM, beta-alanine 17 mM, alpha ketoglutarate 11 mM and pyridoxal phosphate 5 micron.     

Kinetics and thermodynamics of diacetyl reduction with NADPH by alpha-dicarbonyl reductase from pigeon liver

alpha-dicarbonyl reductase from pigeon liver catalyzes diacetyl reduction with NADPH via an ordered Bi-Bi mechanism in which the coenzyme is the leading substrate, as deduced from the inhibition pattern by products and by acetone. The activation energy of the reaction has been calculated as 16.6 kcal/mol, delta H and delta F as 15.6 and 15.3 kcal/mol, respectively, and delta S as 1 cal/mol per k. Kinetic constants obtained for substrates (KmNADPH = 15 microM, KsNADPH = 10 microM; Kmdiacetyl = 0.5 mM, Ksdiacetyl = 0.35 mM) and products (KiNADP 50 microM; Kiacetoin = 100 mM) are about 10 times lower than those reported for this enzyme in the reduction of diacetyl with NADH. This confirms that NADPH is its physiological coenzyme.     

A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports

A number of bacterial lipases can be immobilized in a rapid and strong fashion on octyl-agarose gels (e.g., lipases from Candida antarctica, Pseudomonas fluorescens, Rhizomucor miehei, Humicola lanuginosa, Mucor javanicus, and Rhizopus niveus). Adsorption rates in absence of ammonium sulfate are higher than in its presence, opposite to the observation for typical hydrophobic adsorption of proteins. At 10 mM phosphate, adsorption of lipases is fairly selective allowing enzyme purification associated with their reversible immobilization. Interestingly, these immobilized lipase molecules show a dramatic hyperactivation. For example, lipases from R. niveus, M. miehei, and H. lanuginosa were 6-, 7-, and 20-fold more active than the corresponding soluble enzymes when catalyzing the hydrolysis of a fully soluble substrate (0.4 mM p-nitrophenyl propionate). Even higher hyperactivations and interesting changes in stereospecificity were also observed for the hydrolysis of larger soluble chiral esters (e.g. (R,S)-2-hydroxy-4-phenylbutanoic ethyl ester). These results suggest that lipases recognize these "well-defined" hydrophobic supports as solid interfaces and they become adsorbed through the external areas of the large hydrophobic active centers of their "open and hyperactivated structure". This selective interfacial adsorption of lipases becomes a very promising immobilization method with general application for most lipases. Through this method, we are able to combine, via a single and easily performed adsorption step, the purification, the strong immobilization, and a dramatic hyperactivation of lipases acting in the absence of additional interfaces, (e.g., in aqueous medium with soluble substrate). Copyright 1998 John Wiley & Sons, Inc.     

Enzymatic assay of gamma-cystathionase activity using pyruvate oxidase-peroxidase sequential reaction

A highly sensitive method has been developed for the determination of gamma-cystathionase (EC. 4.4.1.1.) activity in rat tissues using beta-chloro-L-alanine as a substrate. This method is based on colorimetry for the determination of pyruvate produced from beta-chloro-L-alanine with the beta-elimination catalyzed by gamma-cystathionase, coupling a color enzymatic reaction with pyruvate oxidase and peroxidase. The absorbance increases with the oxidized color of a leuco dye, N-(carboxymethylamino)-4,4'-bis (dimethylamino)-diphenylamine at 727 nm is proportional to the gamma-cystathionase activity. The present method is more sensitive and more rapid than the usual methods and does not require troublesome steps such as centrifugation. The calibration curve is linear up to 1.6 microg of partially purified enzyme (100 U/l). Comparison with the usual method with L-homoserine as a substrate gave good correlation (r=0.990). The present method was applied to the determination of gamma-cystathionase activity in adult male rat tissues. The mean activities in liver and kidney were 8.03 and 3.91 U/g wet weight (n=10), respectively.     

Site-directed mutagenesis of the beta subunit of tryptophan synthase from Salmonella typhimurium. Role of active site glutamic acid 350.

To investigate the functional role of glutamic acid 350 in the active site of the beta subunit of tryptophan synthase from Salmonella typhimurium, we have replaced this residue by glutamine or alanine by use of site-directed mutagenesis. The mutant alpha 2 beta 2 complexes were expressed, purified, crystallized, and characterized by spectroscopic and kinetic studies with several substrates. We find large alterations in the substrate and reaction specificity of each mutant form of the alpha 2 beta 2 complex. Since the two mutant enzymes are virtually inactive in reactions with L-serine but are active in reactions with beta-chloro-L-alanine, glutamic acid 350 may facilitate the beta-elimination of the weak hydroxyl leaving group of L-serine. The mutant alpha 2 beta 2 complexes are more active than the wild type enzyme in the beta-elimination reaction with beta-chloro-L-alanine. These enzymes are irreversibly inactivated by beta-chloro-L-alanine, whereas the wild type enzyme is not. These altered properties may result from a change in the conformation of the active site, from a change in the orientation of the coenzyme relative to active site residues, or from a change in the solvent accessibility of the active site. The alteration in the active site may enhance the release of amino acrylate from the Schiff base intermediate by hydrolysis or by transamination.     

Characterization of leukotriene C4 synthetase in mouse peritoneal exudate cells

Cell lysates of mouse peritoneal macrophages, in the presence of reduced glutathione, converted leukotriene LTA4 to LTC4, and neither LTD4 nor LTE4 was detected. Therefore, like cultured rat basophilic leukemia cells (RBL cells), the peritoneal macrophage contains LTC4 synthetase and appears to contain little, if any, gamma-glutamyl transpeptidase. When LTA4 was added to subcellular fractions of mouse macrophage lysate, the highest specific activity of LTC4 synthetase (nmol LTC4/mg protein per 10 min) was associated with the particulate or membrane fractions (i.e., 10(4) and 10(5) X g pellets). The 10(5) X g supernatant contains approx. 1% of the specific activity and 6% of the total LTC4 synthetase activity compared with that of the 10(5) X g pellet. Conversely, the 10(5) X g supernatant had four-times more specific activity and 19-times more total GSH S-transferase activity than did the 10(5) X g pellet when evaluated using 1-chloro-2,4-dinitrobenzene (DNCB) as the substrate. LTA4 was converted to LTC4 by the membrane enzyme LTC4 synthetase in a dose-dependent manner at low LTA4 concentrations (3-50 microM) and reached a plateau of approx. 30 microM LTA4 using the macrophage 10(5) X g pellet as an enzyme source. The apparent Km value of LTC4 synthetase for LTA4 was estimated to be 5 microM based on Lineweaver-Burk plots. Enzyme in the 10(5) X g supernatant produced negligible quantities of LTC4 (1% or less of the particulate fractions) over a wide range of LTA4 concentrations. However, an enzyme in the 10(5) X g supernatant fraction presumed to be GSH S-transferase effectively catalyzes the conjugation of glutathione (GSH) with the aromatic compound DNCB. The apparent Km value of GSH S-transferase for DNCB was estimated to be 1.0-1.5 mM. On the other hand, enzyme from the membrane fraction (i.e., 10(5) X g pellet) catalyzed this reaction at a negligible rate over a wide range of DNCB concentrations. The apparent Km value of LTC4 synthetase for GSH was estimated to be 0.36 mM and the corresponding Km value estimated for the glutathione S-transferase was 0.25-0.76 mM. These values indicate similar kinetics for GSH utilization by both enzymes. These Km values are also significantly lower than the intracellular GSH levels of 2 to 5 mM. Therefore, it is suggested that the substrate limiting LTC4 synthetase activity is LTA4 and not GSH. Our results indicate that LTC4 synthetase from mouse peritoneal macrophages is a particulate or membrane-bound enzyme, as was reported by Bach et al.(ABSTRACT TRUNCATED AT 400 WORDS)     

A new amino acid racemase with threonine alpha-epimerase activity from Pseudomonas putida: purification and characterization.

We have found that Pseudomonas putida ATCC 17642 cells grown in a medium containing D-threonine as the sole nitrogen source produce an enzyme that catalyzes epimerization of threonine. Proton nuclear magnetic resonance analysis of the enzyme reaction in deuterium oxide clearly showed epimerization from L- to D-allo-threonine and also from D- to L-allo-threonine. This is the first example of an enzyme that was clearly shown to epimerize threonine. The enzyme has been purified to homogeneity, which was shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 82,000 and consists of two subunits identical in molecular weight (about 41,000). The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of subunit as a cofactor, and its absorption spectrum exhibits absorption maxima at 280 and 420 nm. The enzyme catalyzes not only epimerization of threonine by stereoconversion at the alpha position but also racemization of various amino acids, except acidic and aromatic amino acids. The enzyme is similar to amino acid racemase with low substrate specificity (EC 5.1.1.10) in enzymological properties but is distinct from it in the action on threonine.     

D-amino acid aminotransferase of Bacillus sphaericus. Enzymologic and spectrometric properties.

D-Amino acid aminotransferase, purified to homogeneity and crystallized from Bacillus sphaericus, has a molecular weight of about 60,000 and consists of two subunits identical in molecular weight (30,000). The enzyme exhibits absorption maxima at 280, 330, and 415 nm, which are independent of the pH (5.5 to 10.0), and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. One of the pyridoxal-5'-P, absorbing at 415 nm, is bound in an aldimine linkage to the epsilon-amino group of a lysine residue of the protein, and is released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive form, which is reactivated by addition of pyridoxal 5'phosphate, still has a 330 nm peak and contains 1 mol of pyridoxal 5'-phosphate. Therefore, this form is regarded as a semiapoenzyme. The holoenzyme shows negative circular dichroic bands at 330 and 415 nm. D-Amino acid aminotransferase catalyzes alpha transamination of various D-amino acids and alpha-keto acids. D-Alanine, D-alpha-aminobutyrate and D-glutamate, and alpha-ketoglutarate, pyruvate, and alpha-ketobutyrate are the preferred amino donors and acceptors, respectively. The enzyme activity is significantly affected by both the carbonyl and sulfhydryl reagents. The Michaelis constants are as follows: D-alanine (1.3 and 4.2 mM with alpha-ketobutyrate and alpha-ketoglutarate, respictively), alpha-ketobutyrate (14 mM withD-alanine), alpha-ketoglutarate (3.4 mM with D-alanine), pyridoxal 5'-phosphate (2.3 muM) and pyridoxamine 5'-phosphate (25 muM).     

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤50°C) was 10°C higher than that for CITase-T3040 (≤40°C); the k(cat)/K(M) value of CITase-598K was approximately two times higher (32.2s(-1)mM(-1)) than that of CITase-T3040 (17.8s(-1)mM(-1)). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.     

Enzymatic formation of uridine diphosphate N-acetyl-D-mannosamine.

An enzyme that catalyzes the interconversion of UDP-N-acetyl-D-glucosamine and UDP-N-acetyl-D-mannosamine was purified about 700-fold from the supernatant fraction of Bacillus cereus, and the properties of this enzyme were studied. This enzyme was not stimulated by NAD+, NADH, or any metal ions. The optimum pH was between 7.5 and 8.0. At equilibrium of the reaction, the ratio of UDP-N-acetylglucosamine to UDP-N-acetylmannosmaine was about 9:1. The enzyme was inactive toward free N-acetylhexosamines, their phosphate esters, UDP-glucose, and UDP-N-acetylgalactosamine. A stimulatory role of UDP-N-acetylglucosamine was demonstrated. In the reaction with UDP-N-acetylglucosamine, the rate as a function of substrate concentration showed a sigmoidal relationship with a Hill coefficient of 1.8 and an apparent Km value for UDP-N-acetylglucosamine of 1.1 mM. The reverse reaction with UDP-N-acetylmannosamine required the presence of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine concentration required for half-maximal activation was about 0.5 mM. The apparent Km for UDP-N-acetylmannosamine measured in the presence of 0.5 mM UDP-N-acetylglucosamine was 0.22mM. Other nucleotides or hexosamine derivatives were not stimulatory. The same activity was found in cell extracts from several bacterial species.     

Rapid purification and biochemical characterization of glucose kinase from Streptomyces peucetius var. caesius

Glucose kinase catalyzes the ATP-dependent phosphorylation of glucose. Streptomyces peucetius var. caesius glucose kinase was purified 292-fold to homogeneity. The enzyme has cytosolic localization and is composed of four identical subunits, each of 31 kDa. The purified enzyme easily dissociates into dimers. However, in the presence of 100 mM glucose the enzyme maintains its tetrameric form. Maximum activity was found at 42 degrees C and pH 7.5. Isoelectric focusing of the enzyme showed a pl of 8.4. The N- and C-terminal amino acid sequences were MGLTIGVD and VYFAREPDPIM, respectively. The kinetic mechanism of S. peucetius var. caesius glucose kinase appears to be a rapid equilibrium ordered type, i.e., ordered addition of substrates to the enzyme, where the first substrate is d-glucose. The K(m) values for d-glucose and MgATP(2-) were 1.6 +/- 0.2 and 0.8 +/- 0.1 mM, respectively. Mg(2+) in excess of 10 mM inhibits enzyme activity.     

5-Azidoindole binding to the Escherichia coli tryptophan synthase alpha 2 beta 2 complex

The tryptophan synthase alpha 2 beta 2 complex catalyzes tryptophan (Trp) biosynthesis from serine plus either indole (IN) or indole-3-glycerol phosphate (InGP). The photoreactive 5-azido analog in IN (AzIN), itself a substrate in the dark, was utilized to examine the substrate binding sites on this enzyme. When irradiated with AzIN at concentrations approaching IN saturation for the IN----Trp activity (0.1 mM), in the absence of serine, the enzyme was increasingly inactivated (up to 70-80%) concomitant with the progressive binding of a net of 2 mol AzIN per alpha beta equivalent. Little or no cooperativity in the binding of the 2 mol AzIN was observed. In contrast, there was minimal effect on the IN----InGP activity. Under these conditions AzIN appeared to be incorporated equally into each subunit. No significant inactivation nor binding occurred in the presence of serine. A quantitatively similar inactivation of InGP----Trp activity was observed over the same AzIN concentration range, suggesting common IN sites for Trp biosynthesis from either indole substrate. At higher concentrations (0.1-0.7 mM), no further inactivation occurred, although there was extensive additional binding (up to 10 mol/alpha beta equivalent). These data are consistent, although more clear-cut quantitatively, with the high- and low-affinity sites proposed from equilibrium dialysis studies. AzIN binding studies utilizing the isolated beta 2 subunit confirmed earlier reports suggesting the existence of many nonspecific IN binding sites on this subunit.     

Bacterial N-succinyl-L-diaminopimelic acid desuccinylase. Purification, partial characterization, and substrate specificity

The enzyme N-succinyl-L-diaminopimelic acid desuccinylase from Escherichia coli has been purified 7,100-fold to apparent homogeneity. The enzyme is part of the diaminopimelic acid-lysine pathway in bacteria and catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to produce L-diaminopimelic acid and succinate. The enzyme exists as a mixture of dimeric and tetrameric species of identical subunits of molecular weight approximately 40,000. Activity was completely abolished following dialysis of the enzyme against metal chelators. Cobalt(II) and zinc were effective in restoring the activity. The apparent affinities of the apoenzyme for cobalt and zinc were similar (Kd values near 1 microM) and the cobalt enzyme was 2.2-fold more active than the zinc enzyme. The Km and turnover number for the hydrolysis of the natural substrate, N-succinyl-L-diaminopimelic acid, were 0.4 mM and 16,000 min-1, respectively. The substrate specificity of the enzyme was defined by preparing a number of substrate analogues that systematically lack the various functional groups present in the molecule. These studies show that the enzyme is highly specific for the natural substrate. These properties of N-succinyl-L-diaminopimelic acid desuccinylase and the fact that the enzyme is essential for bacterial growth make it an ideal target for the development of inhibitors with potential antibacterial activity.