Heterologous expression and characterization of mouse spermine oxidase

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.     

Tyrosine-phosphorylated caveolin is a physiological substrate of the low M(r) protein-tyrosine phosphatase

Low M(r) phosphotyrosine-protein phosphatase is involved in the regulation of several tyrosine kinase growth factor receptors. The best characterized action of this enzyme is on the signaling pathways activated by platelet-derived growth factor, where it plays multiple roles. In this study we identify tyrosine-phosphorylated caveolin as a new potential substrate for low M(r) phosphotyrosine-protein phosphatase. Caveolin is tyrosine-phosphorylated in vivo by Src kinases, recruits into caveolae, and hence regulates the activities of several proteins involved in cellular signaling cascades. Our results demonstrate that caveolin and low M(r) phosphotyrosine-protein phosphatase coimmunoprecipitate from cell lysates, and that a fraction of the enzyme localizes in caveolae. Furthermore, in a cell line sensitive to insulin, the overexpression of the C12S dominant negative mutant of low M(r) phosphotyrosine-protein phosphatase (a form lacking activity but able to bind substrates) causes the enhancement of tyrosine-phosphorylated caveolin. Insulin stimulation of these cells induces a strong increase of caveolin phosphorylation. The localization of low M(r) phosphotyrosine-protein phosphatase in caveolae, the in vivo interaction between this enzyme and caveolin, and the capacity of this enzyme to rapidly dephosphorylate phosphocaveolin, all indicate that tyrosine-phosphorylated caveolin is a relevant substrate for this phosphatase.     

Production and Characterization of Laccase from Botrytis cinerea 61-34

An isolate of Botrytis cinerea (strain 61-34) constitutively expresses substantial amounts of extracellular laccase on a defined growth medium. The enzyme has been purified to homogeneity by a facile operational sequence, the last stage of which involves hydrophobic interaction chromatography. By these means, over 80 mg of laccase liter(sup-1) can be obtained from aerated fermentor reaction broths. The enzyme, with an estimated M(infr) of 74,000 and pI of 4.0, is a monomeric glycoprotein containing 49% carbohydrate predominantly as hexose. With 2,6-dimethoxyphenol, it exhibits a pH optimum of 3.5 and a temperature optimum of 60(deg)C, and its K(infm) is 100 (mu)M. The purified enzyme with this substrate has a specific activity of 9.1 mkat mg of protein(sup-1). Taken together with a broad substrate range and its stability in 4% sodium dodecyl sulfate or 2 M urea solutions, several biotechnology transfers are suggested.     

Cloning and sequence analysis of the gene encoding an NADP-dependent alcohol dehydrogenase in Mycobacterium bovis BCG.

The nucleotide sequence of a 1619-bp fragment of Mycobacterium bovis BCG containing the gene that encodes an alcohol dehydrogenase (ADH) has been determined. The M(r) calculated from the deduced amino acid (aa) sequence, as well as the N terminus, are in good accordance with those determined for the ADH purified from M. bovis BCG extracts. The M. bovis BCG cloned adh gene was expressed in Escherichia coli by its own promoter and the synthesized product shows ADH activity in the butane-1-ol-NADP system. Based on comparison of the aa sequence, this enzyme belongs to the zinc-containing, long-chain alcohol/polyol dehydrogenase family, which has been primarily described in eukaryotes. Of the 22 strictly conserved residues in this group, 19 are also conserved in M. bovis BCG ADH (BCGADH).     

A beta-lysine adenylating enzyme and a beta-lysine binding protein involved in poly beta-lysine chain assembly in nourseothricin synthesis in Streptomyces noursei.

Nourseothricins (syn. Streptothricins), a group of nucleoside peptides produced by several streptomycete strains, contain a poly beta-lysine chain of variable length attached in amide linkage to the amino sugar moiety gulosamine of the nucleoside portion. We show that the nourseothricin-producing Streptomyces noursei contains an enzyme (NpsA) of an apparent M(r) 56,000 that specifically activates beta-lysine by adenylation but does not bind to it as a thioester. Cloning and sequencing of npsA from S. noursei including its flanking DNA regions revealed that it is closely linked to the nourseothricin resistance gene nat1 and some other genes on the chromosome possibly involved in nourseothricin biosynthesis. The deduced amino-acid sequence revealed that NpsA is a stand-alone adenylation domain with similarity to the adenylation domains of nonribosomal peptide synthetases (NRPS). Further analysis revealed that S. noursei contains a beta-lysine binding enzyme (NpsB) of about M(r) 64,100 which can be loaded by NpsA with beta-lysine as a thioester. Analysis of the deduced amino-acid sequence from the gene (npsB) of NpsB showed that it consists of two domains. The N-terminal domain of approximately 100 amino-acid residues has high similarity to PCP domains of NRPSs whereas the 450-amino-acid C-terminal domain has a high similarity to epimerization (E)-domains of NRPSs. Remarkably, in this E-domain the conserved H-H-motif is changed to H-Q, which suggests that either the domain is nonfunctional or has a specialized function. The presence of one single adenylating beta-lysine activating enzyme in nourseothricin-producing streptomycete and a separate binding protein suggests an iteratively operating NRPS-module catalyses synthesis of the poly beta-lysine chain.     

Cathepsin M: a lysosomal proteinase with aldolase-inactivating activity

A proteinase, designated cathepsin M, that catalyzes the limited modification and inactivation of fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) and fructose 1,6-bisphosphatase (EC 3.1.3.11) has been partially purified from rabbit liver. On the basis of its molecular size (M_r = 30,000), activation by sulfhydryl compounds and inhibition by leupeptin it has been characterized as a B-type cathepsin, but other properties distinguish it from cathepsins B, L, and H. Approximately 50% of the total cathepsin M activity is associated with membranes prepared from disrupted lysosomes; this fraction of the activity is also expressed by intact lysosomes. In the membrane-bound form the enzyme is active at neutral pH, but the soluble enzyme and the activity eluted from the membranes are maximally active at pH 5.0. Fasting increases the activity of cathepsin M; the increase is almost entirely in the membrane-bound fraction.     

Inhibition by substrate of fructose 1,6-bisphosphatase purified from rat kidney cortex. Calculation of the kinetic constants of the enzyme

Fructose 1,6-bisphosphatase is a typical enzyme that is severely inhibited by its own substrate. This makes it difficult to determine all the parameters involved in its kinetics. It has been shown recently that if Vm is satisfactorily estimated the remaining parameters can be determined using the Hill plot (Bounias, M. (1988) Biochem. Int. 17, 147-154). The enzyme has been purified from rat kidney cortex nearly to homogeneity, and its kinetic constants have been calculated using a rigorous algebraic method. The most interesting result is that the substrate is unable to bind to the free enzyme as an inhibitor, which indicates that the enzyme lacks an allosteric site for hexose bisphosphates.     

Inhibition and affinity chromatography of chicken lung angiotensin I-converting enzyme with captopril.

1. Angiotensin I-converting enzyme (EC 3.4.15.1) has been purified to electrophoretic homogeneity from chicken lung by using a facile two-step protocol which included affinity chromatography on Sepharose-bound captopril. 2. Captopril was a potent inhibitor of chicken lung angiotensin I-converting enzyme with Ki values of 2.0 nmol/l and 1.6 nmol/l for detergent-extracted and trypsin-extracted angiotensin I-converting enzymes, respectively. 3. Molecular weight comparison of trypsin-extracted (M(r)270,000) and detergent-extracted (M(r)690,000) angiotensin I-converting enzyme indicated that membrane-binding sequence contributed to a large extent to the enzyme molecule. 4. Kinetic properties of both forms of the enzyme suggested that the membrane-bound sequence contributed to an increase of the enzyme-substrate affinity.     

Characterization of changes in the activity of K+-n-nitrophenylphosphatase of erythrocyte ghosts under the influence of ouabain

Variation of K+-p-NPP-ase of human ghosts under the action of ouabain (10(-11)-10(-3) M) has been studied. In human ghosts the activity of ouabain-sensitive K+-p-NPP-ase has been found to make up 65% of the total enzyme activity. The activity of ouabain-sensitive K+-p-NPP-ase reaches a maximum level at pH 7.6-8.0. A decrease in the activity of this enzyme caused by ouabain is of two-phase character. In the range of ouabain concentration from 10(-10) M to 10(-6) M and from 10(-5) M to 10(-3) M the enzyme activity lowers significantly; in the range of 10(-7) M to 10(-5) M it reaches the plato. Two types of the enzyme are assumed to exist differing by 4-5 orders of magnitude in their sensitivity to ouabain, inhibitor affinity constants and Michaelis constants.     

A NAD(P)H oxidase isolated from the archaeon Sulfolobus solfataricus is not homologous with another NADH oxidase present in the same microorganism. Biochemical characterization of the enzyme and cloning of the encoding gene

A NAD(P)H oxidase has been isolated from the archaeon Sulfolobus solfataricus. The enzyme is a homodimer with M(r) 38,000 per subunit (SsNOX38) containing 1 FAD molecule/subunit. It oxidizes NADH and, less efficiently, NADPH with the formation of hydrogen peroxide. The enzyme was resistant against chemical and physical denaturating agents. The temperature for its half-denaturation was 93 and 75 degrees C in the absence or presence, respectively, of 8 M urea. The enzyme did not show any reductase activity. The SsNOX38 encoding gene was cloned and sequenced. It accounted for a product of 36.5 kDa. The translated amino acid sequence was made of 332 residues containing two putative betaalphabeta-fold regions, typical of NAD- and FAD-binding proteins. The primary structure of SsNOX38 did not show any homology with the N-terminal amino acid sequence of a NADH oxidase previously isolated from S. solfataricus (SsNOX35) (Masullo, M., Raimo, G., Dello Russo, A., Bocchini, V. and Bannister, J. V. (1996) Biotechnol. Appl. Biochem. 23, 47-54). Conversely, it showed 40% sequence identity with a putative thioredoxin reductase from Bacillus subtilis, but it did not contain cysteines, which are essential for the activity of the reductase.     

Dinitrophenyl S-glutathione ATPase purified from human muscle catalyzes ATP hydrolysis in the presence of leukotrienes.

Dinitrophenyl S-glutathione (Dnp-SG) ATPase has been purified from human muscle to apparent homogeneity using Dnp-SG affinity chromatography and immunoaffinity chromatography using antibodies raised against human erythrocyte Dnp-SG ATPase. The enzyme purified from human muscle showed a subunit M(r) value of about 38 kDa in denaturing gels. The M(r) value of the native enzyme as determined by Sephadex G-200 gel filtration was found to be about 80 kDa, which indicates that it is a dimer. The N-terminus of the enzyme was blocked. Its immunological and kinetic properties were similar to Dnp-SG ATPase of human erythrocytes. Besides catalyzing the ATP hydrolysis in the presence of Dnp-SG, the muscle enzyme also catalyzed ATP hydrolysis in the presence of various leukotrienes, namely LTC4.LTD4, LTE4, and N-acetyl LTE4. The specific activity of the enzyme toward LTC4 was relatively higher than other GSH-xenobiotic conjugates. The muscle enzyme exhibits a low Km value for all leukotrienes as compared to Dnp-SG, indicating high affinity of the enzyme for leukotrienes as activators. The enzyme also catalyzed ATP hydrolysis in the presence of GSH conjugates of endogenously generated fatty acid epoxides. Our results might suggest that Dnp-SG ATPase is involved in the transport of GSH conjugates, leukotrienes, and other organic anions in muscle, erythrocytes, liver, and probably other tissues.     

Acetylornithine deacetylase, succinyldiaminopimelate desuccinylase and carboxypeptidase G2 are evolutionarily related.

The nucleotide (nt) sequence of the Escherichia coli argE gene, encoding the acetylornithine deacetylase (AO) subunit, has been established and corresponds to a 43-kDa (M(r) 42,320) polypeptide. The enzyme has been purified to near homogeneity and it appears to be a dimer consisting of two 43-kDa subunits. The amino acid sequence deduced from the nt sequence was compared to that of the subunit of E. coli succinyldiaminopimelate desuccinylase (the dapE gene product involved in the diaminopimelate pathway for lysine biosynthesis), since both enzymes share functional and biochemical features. Significant similarity covering the entire sequence allows us to infer a common origin for both deacylases. This homology extends to the Pseudomonas sp. G2 carboxypeptidase (G2CP); this or a functionally related enzyme may be responsible for the minor AO activity found in organisms relying on ornithine acetyltransferase for ornithine biosynthesis.     

Synthesis of a novel photoaffinity derivative of 1-deoxynojirimycin for active site-directed labeling of glucosidase I

Glucosidase I releases the distal alpha1,2-glucosyl residue in the Glc(3)Man(9)GlcNAc(2) precursor immediately after its transfer from the dolichol-P-P-linked intermediate in the endoplasmic reticulum and triggers the processes for the posttranslational remodeling, folding, and maturation of N-linked glycoproteins. The enzyme has been purified and characterized from several eukaryotic systems. Its cDNA and the gene have also been cloned. The enzyme is a target for the development of drugs for several pathological conditions. A structural analysis on the biochemically purified enzyme has been hampered because of its low abundance and unstable character. The recombinant enzyme has not been obtained in quantity and characterized. Glucosidase I is strongly inhibited by the glucose analog 1-deoxynojirimycin (DNM). To gain an insight into the architecture of the active site of the enzyme, we here report the synthesis of a photoactive derivative of DNM, viz. 4-(rho-azidosalicylamido)butyl-5-amido-pentyl-1-DNM (ASBA-P-DNM). With an IC(50) of 0.42 micro M, it is nearly nine times stronger inhibitor than DNM (IC(50) = 3.5 micro M). On photolysis, the bound [(125)I]ASBA-P-DNM specifically labels the native enzyme, which yields a 24-kDa peptide after treatment with V8 protease, apparently representing the region around its active site. Thus ASBA-P-DNM should serve as a novel reagent to conduct structure-function analysis on glucosidase I.     

Glutathione synthesis in Streptococcus agalactiae. One protein accounts for gamma-glutamylcysteine synthetase and glutathione synthetase activities

Gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS), distinct enzymes that together account for glutathione (GSH) synthesis, have been isolated and characterized from several Gram-negative prokaryotes and from numerous eukaryotes including mammals, amphibians, plants, yeast, and protozoa. Glutathione synthesis is relatively uncommon among the Gram-positive bacteria, and, to date, neither the genes nor the proteins involved have been identified. In the present report, we show that crude extracts of Streptococcus agalactiae catalyze the gamma-GCS and GS reactions and can synthesize GSH from its constituent amino acids. The putative gene for S. agalactiae gamma-GCS was identified and cloned, and the corresponding protein was expressed and purified. Surprisingly, it was found that the isolated enzyme catalyzes both the ATP-dependent synthesis of L-gamma-glutamyl-L-cysteine from L-glutamate and L-cysteine and the ATP-dependent synthesis of GSH from L-gamma-glutamyl-L-cysteine and glycine. This novel bifunctional enzyme, referred to as gamma-GCS-GS, has been characterized in terms of catalytic activity, substrate specificity, and inhibition by GSH, cystamine, and transition state analog sulfoximines. The N-terminal 518 amino acids of gamma-GCS-GS (total M(r) 85,000) show 32% identity and 43% similarity with E. coli gamma-GCS (M(r) 58,000), but the C-terminal putative GS domain (remaining 202 amino acids) of gamma-GCS-GS shows no significant homology with known GS sequences. The C terminus (360 amino acids) is, however, homologous to D-Ala, D-Ala ligase (24% identity; 38% similarity), an enzyme having the same protein fold as known GS proteins. These results are discussed in terms of the evolution of GSH synthesis and the possible occurrence of a similar bifunctional GSH synthesis enzyme in other bacterial species.     

Novel heme-containing lyase, phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1: purification, characterization, and molecular cloning of the gene

A novel dehydratase that catalyzes the stoichiometric dehydration of Z-phenylacetaldoxime to phenylacetonitrile has been purified 483-fold to homogeneity from a cell-free extract of Bacillus sp. strain OxB-1 isolated from soil. It has a M(r) of about 40 000 and is composed of a single polypeptide chain with a loosely bound protoheme IX. The enzyme is inactive unless FMN is added to the assay, but low activity is also observed when sulfite replaces FMN. The activity in the presence of FMN is enhanced 5-fold under anaerobic conditions compared to the activity measured in air. The enzyme has maximum activity at pH 7.0 and 30 degrees C, and it is stable at up to 45 degrees C at around neutral pH. The aerobically measured activity in the presence of FMN is also enhanced by Fe(2+), Sn(2+), SO(3)(2)(-), and NaN(3). Metal-chelating reagents, carbonyl reagents, electron donors, and ferri- and ferrocyanides strongly inhibit the enzyme with K(i) values in the micromolar range. The enzyme is active with arylalkylaldoximes and to a lesser extent with alkylaldoximes. The enzyme prefers the Z-form of phenylacetaldoxime over its E-isomer. On the basis of its substrate specificity, the enzyme has been tentatively named phenylacetaldoxime dehydratase. The gene coding for the enzyme was cloned into plasmid pUC18, and a 1053 base-pair open reading frame that codes for 351 amino acid residues was identified as the oxd gene. A nitrilase, which participates in aldoxime metabolism in the organism, was found to be coded by the region just upstream from the oxd gene. In addition an open reading frame (orf2), whose gene product is similar to bacterial regulatory (DNA-binding) proteins, was found just upstream from the coding region of the nitrilase. These findings provide genetic evidence for a novel gene cluster that is responsible for aldoxime metabolism in this microorganism.     

An octameric prokaryotic glutamine synthetase from the haloarchaeon Haloferax mediterranei

The glutamine synthetase (EC 6.3.1.2) from the haloarchaeon Haloferax mediterranei has been purified and characterized in order to understand the ammonium assimilation in haloarchaea. Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography, the enzyme consists of eight subunits of 51.7 kDa, suggesting that this enzyme belongs to the glutamine synthetase type II. The purified enzyme has been characterized with respect to its optimum temperature (45 degrees C) and pH value (8.0). The optimal NaCl or KCl concentrations for the reaction were 0.5 and 0.25 M, respectively. The effect of l-methionine-d, l-sulphoximine and different divalent metal ions has also been tested. The glutamine synthetase presented here is unusual; it shows the typical characteristic of eukaryotic and soil bacteria glutamine synthetases.     

Properties of 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'- phosphate reductase, a enzyme of the second stage of flavinogenesis in Pichia guilliermondii yeasts

2,5-Diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been isolated from cells of Pichia guilliermondii and subjected to 20-fold purification by treating extracts with streptomycin sulphate, frationating proteins (NH4)2SO4 at 45-75% of saturation and chromatography on blue sepharose CL-6B. The use of gel filtration through Sephadex G-150 and chromatography on DEAE-cellulose proved to be less effective for the enzyme purification. It has been established that it is 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5-phosphate but not its dephosphorylated form that is the substrate of the given reductase; Km is equal to 7.10(-5) M. The reaction proceeds in the presence of NADPH or NADH. The enzyme affinity to NADPH (Km = 4.7.10(-5) M) is approximately one order higher than that to NADPH (Km = 5.5.10(-4) M). The enzyme manifests the optimum of action at pH 7.2 and the temperature of 37 degrees C; the molecular weight is 140 kD. EDTA as well as flavins in the concentration of 1.10(-3) M exert no effect on the reductase activity. The enzyme is labile at 4 degrees C and is inactivated in the frozen state at -15 degrees C. The 2.5-diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been also revealed in Torulopsis candida, Debaryomyces kl?ckeri, Schwanniomyces occidentalis, Eremothecium ashbyii (flavinogenic species) and Candida utilis. Aspergillus nidulans, Neurospora crassa (nonflavinogenic species). The synthesis of this enzyme contrary to other enzymes of the riboflavin biosynthesis is not regulated in flavinogenic yeast by iron ions.