Sequence and homology model of 3-isopropylmalate dehydrogenase from the psychrotrophic bacterium Vibrio sp. I5 suggest reasons for thermal instability

The leuB gene from the psychrotrophic strain Vibrio sp. I5 has been cloned and sequenced. The gene codes for 3-isopropylmalate dehydrogenase, a 360-residue, dimeric enzyme involved in the biosynthesis of leucine. Three recently solved homologous isopropylmalate dehydrogenase (IPMDH) crystal structures from thermophilic and mesophilic organisms have been used to build a homology model for the psychrotrophic IPMDH and to deduce the possible structural reasons for its decreased thermostability. According to our model the psychrotrophic IPMDH contains fewer stabilizing interactions than its mesophilic and thermophilic counterparts. Elements that have been identified as destabilizing in the comparison of the psychrotrophic, mesophilic and thermophilic IPMDHs are a smaller number of salt-bridges, a reduction in aromatic-aromatic interactions, fewer proline residues and longer surface loops. In addition, there are a number of substitutions of otherwise strictly conserved residues that can be linked to thermostability.     

Nucleoside 2-deoxyribosyltransferase. Pre-steady-state kinetic analysis of native enzyme and mutant enzyme with an alanyl residue replacing Glu-98

Nucleoside 2-deoxyribosyltransferase catalyzes cleavage of a 2'-deoxyribosylnucleoside (A) to a nucleobase (P) with deoxyribosylation of the enzyme. Substrates quenched the intrinsic fluorescence of native enzyme (E) and a catalytically inactive mutant enzyme (E98A enzyme). The time courses of these reactions were analyzed in terms of the following scheme where EX is the 2-deoxyribosyl ester of Glu-98. [formula: see text] The initial complexes between E and dAdo, dGuo, dIno, and dCyd or those between EX and the corresponding nucleobases were formed in a rapid equilibrium step. Native enzyme and E98A enzyme bound 2'-deoxyribosylnucleosides with similar affinities (k-1/k1). From a comparison of the time-dependent fluorescence changes associated with the reaction of native enzyme or E98A enzyme with these substrate, the kinetic step for 2-deoxyribosylation of Glu-98 was identified (k2 and k-2). dThd and dUrd quenched the fluorescence of native enzyme in a biphasic process. The late phase of this reaction was associated with 2-deoxyribosylation of Glu-98. The pre-steady-state kinetic constants calculated from fluorescence quenching data for dAdo and Cyt were consistent with the experimental values for the steady-state kinetic coefficients and the equilibrium constant of the reaction.     

The conversion from the dehydrogenase type to the oxidase type of rat liver xanthine dehydrogenase by modification of cysteine residues with fluorodinitrobenzene

When rat liver xanthine dehydrogenase was incubated with fluorodinitrobenzene (FDNB) at pH 8.5, the total enzyme activity decreased gradually to a limited value of initial activity with modification of two lysine residues in a similar way to the modification of bovine milk xanthine oxidase with FDNB (Nishino, T., Tsushima, K., Hille, R. and Massey, V. (1982) J. Biol. Chem. 257, 7348-7353). After modification with FDNB, the two peptides containing dinitrophenyl-lysine were isolated from the molybdopterin domain after proteolytic digestion and were identified as Lys754 and Lys771 by sequencing the peptides. During the modification of these lysine residues, xanthine dehydrogenase was found to be converted to an oxidase form in the early stage of incubation. Incorporation of the 3H-dinitrophenyl group into enzyme cysteine residues was 0.96 mol per enzyme FAD for 68% conversion to the oxidase form. The modified enzyme was reconverted to the dehydrogenase form by incubation with dithiothreitol with concomitant release of 3H-dinitrophenyl compounds. After modification with 3H-FDNB followed by carboxymethylation under denaturating conditions, the enzyme was digested with proteases. Three 3H-dinitrophenyl-labeled peptides were isolated and sequenced. The modified residues were identified to be Cys535, Cys992 and Cys1324. These residues are conserved among the all known mammalian enzymes, but Cys992 and Cys1324 are not conserved in the chicken enzyme. Cys1324 of the rat enzyme was found not to be involved in the conversion from the dehydrogenase to the oxidase by limited proteolysis experiments, but Cys535 and Cys992 which seemed to be modified alternatively with FDNB appear to be involved in the conversion.     

L-threonine dehydrogenase. Purification and properties of the homogeneous enzyme from Escherichia coli K-12

L-Threonine dehydrogenase, which catalyzes the conversion of L-threonine to aminoacetone + CO2 presumably via the intermediate formation of alpha-amino-beta-ketobutyrate, has been purified to apparent homogeneity from extracts of a mutant of Escherichia coli K-12 which has constitutively derepressed levels of the enzyme. Three fractionation steps were used including controlled heat denaturation, DEAE-Sephadex chromatography, and blue dextran-Sepharose affinity chromatography. The purified enzyme migrated as a single band, coincident with dehydrogenase activity, when electrophoresed on polyacrylamide gels at pH 8.0 and 9.5. Electrophoresis in 1% sodium dodecyl sulfate also showed one band and a single schlieren peak was seen during sedimentation velocity centrifugation. The enzyme has an apparent molecular weight of 140,000 +/- 4,000 as determined by sucrose density and sedimentation equilibrium centrifugation. Based on electrophoresis in 1% sodium dodecyl sulfate, sedimentation equilibrium centrifugation in 6 M guanidine.HCl, and cross-linking with dimethyl suberimidate, the molecule is a tetramer consisting of identical (or nearly identical) subunits with Mr approximately equal to 35,000. L-Threonine dehydrogenase is specific for NAD+ or NAD+ analogs and utilizes L-threonine, D-allothreonine, or L-threonine amide as the best substrates. In 50 mM Tris.HCl buffer (pH 8.4) and 37 degrees C, the Km values for L-threonine and NAD+ are 1.43 and 0.19 mM, respectively. The enzyme has a pH optimum of 10.3, is activated by Mn2+, and shows a substantial loss of activity when treated with certain sulfhydryl-reacting reagents.     

The pyruvate dehydrogenase complex from thermophilic organisms: thermal stability and re-association from the enzyme components

Parabuthus transvaalicus, P. granulatus, and P. villosus are three medically important scorpion species occurring in southern Africa which can cause severe envenoming among people. In contrast to many other genera, no data is available on the venom composition of scorpions belonging to the genus Parabuthus. Here we have investigated the components which may contribute to the venomous potential. The constancy of venom composition within each of the three species and between the three species was investigated by means of gel filtration chromatography. The venoms of the three species each were characterized by a constant and typical elution pattern, resulting in a 'gel filtration fingerprint' which allows distinction between each species. It appears that certain components in the venoms are common to either all three species, or to two of the three species. This points to a clear interspecies relationship within the genus. We also describe the isolation and characterization of some of the polypeptide toxins present in the venoms of P. villosus, P. transvaalicus and P. granulatus by means of reversed phase chromatography and screening of the toxic components on voltage-activated potassium and sodium channels. Our results confirm that toxins which inhibit potassium channels and alter sodium channel gating are present in the venoms studied.     

Carbamate kinase from Enterococcus faecalis and Enterococcus faecium--cloning of the genes, studies on the enzyme expressed in Escherichia coli, and sequence similarity with N-acetyl-L-glutamate kinase

Carbamate kinase (CK) catalyzes the reversible reaction NH2COO- + ATP <--> NHCOOPO3(2-) + ADP, serving to synthesize ATP from carbamoyl phosphate in those microorganisms that derive energy from anaerobic arginine degradation via the arginine dihydrolase pathway. We report here the cloning and sequencing of the CK gene from Enterococcus faecalis and Enterococcus faecium and we demonstrate that the amino acid sequence of CK is identical in the two species. The enzyme, expressed and isolated from Escherichia coli using simple purification procedures, was used to generate crystals suitable for X-ray studies and to investigate the utilization by CK of bicarbonate and other carbamate analogs. CK had a bicarbonate-dependent ATPase activity and, therefore, is able to synthesize carboxyphosphate, an unstable compound that is an intermediate in the reactions catalyzed by carbamoyl-phosphate synthetase (CPS) and by biotin carboxylase. Other functional similarities with CPS include the utilization of acetate by CK with a similarly high Km and the similar Km values of CK for carbamate and of CPS for bicarbonate. Enterococcal CK was inhibited by adenosine(5')pentaphospho(5')adenosine (Ap5A) and Ap6A and, less powerfully, by Ap4A, whereas Ap3A is essentially non-inhibitory. Thus, inhibition by Ap5A seems not to be a valid criterion to differentiate between CK and CPS, for the two enzymes can be inhibited by Ap5A. All these results support the relatedness of CK and CPS. Finally, we used limited proteolysis: (a) to localize the epitopes for monoclonal antibodies obtained against CK; (b) to demonstrate the importance of the C-terminus for enzyme activity; and (c) to show that Arg158 is highly exposed and may be essential for activity. Comparison of the sequence of CK with known protein sequences demonstrates considerable similarity of CK with bacterial N-acetylglutamate kinases, strongly suggesting that these two enzymes may share a similar structure and the same catalytic mechanism.     

Sequence analysis of functional regions of homoserine dehydrogenase genes from L-lysine and L-threonine-producing mutants of Brevibacterium lactofermentum

The homoserine dehydrogenase (HD) genes from Brevibacterium lactofermentum lysine- and threonine-producing mutants were cloned, using the polymerase chain reaction, and sequenced. We found the amino acid substitutions, Val104Ile in the lysine-producing mutants in which HD may cause leaky mutation and Ser393Phe in the threonine-producing mutant with feedback-insensitive HD.     

NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax. The first identified archaeal member of the aldehyde dehydrogenase superfamily is a glycolytic enzyme with unusual regulatory properties

The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The coding gene was cloned, sequenced, and expressed in Escherichia coli. Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases. The activity of the enzyme is affected by a series of metabolites. All effectors tested influence the affinity of the enzyme for its cosubstrate NAD+. Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD+. Additionally, most of the effectors investigated induce cooperativity of NAD+ binding. The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T. tenax. Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the Embden-Meyerhof-Parnas pathway.     

A kinetic analysis of the oligonucleotide-modulated ATPase activity of the helicase domain of the NS3 protein from hepatitis C virus. The first cycle of interaction of ATP with the enzyme is unique

Hepatitis C virus (HCV) helicase (E) formed spectrofluorometrically detectable complexes with a 16-mer and HF16 (a 16-mer with 5'-hexachlorofluoresceinyl moiety). The interaction of helicase with these effectors was investigated by kinetic techniques to determine if the complexes were kinetically competent for ATP hydrolysis. kcat values with the 16-mer and HF16 were 2.7 and 36 s-1, respectively. The maximal value of the rate constant for the approach of an intermediate to the steady-state level has to be at least 4-fold greater than kcat for it to be kinetically competent. This value was 1.2 s-1 with HF16 and "E.ATP" and was 1.82 s-1 with ATP and E.HF16. These values were too small for formation of these intermediates to be kinetically competent in ATP hydrolysis. Dissociation of "E.HF16. ATP" (0.34 s-1) was also too slow to contribute significantly to catalysis. Furthermore, the Km of E.HF16 for ATP (3 mircoM) was significantly less than the Km for ATP hydrolysis at a saturating concentration of HF16 (320 microM). HCV helicase has two nucleotide-binding sites per monomer. If the fluorescence changes observed were associated with structure changes preceding steady-state catalysis (isomerization), pre-steady-state data could be reconciled with the turnover data. Data for the 16-mer yielded similar conclusions.     

Purification and characterization of a new enzyme, N-alkylglycine oxidase from Cladosporium sp. G-10

A new enzyme, N-alkylglycine oxidase, was isolated from a soil mold, Cladosporium sp. G-10. This protein, which was purified to near homogeneity by ammonium sulfate precipitation followed by successive column chromatography on phenyl-Sepharose, DEAE-Sepharose and Sephadex G-200, was a single polypeptide with a molecular mass of 52,000. In the presence of O2 and H2O, this enzyme acted on some N-alkylglycine derivatives, such as N epsilon-carboxymethyllysine, N-carboxymethyl-6-aminocaproic acid, sarcosine and N-ethylglycine, and produced corresponding N-alkylamine, glyoxylic acid and H2O2. This enzyme had optimum activity at 30 degrees C, pH 8-10, and was most inhibited by ZnSO4, pCMB, iodoacetic acid, and SDS.     

Mechanism of malic enzyme from pigeon liver. Magnetic resonance and kinetic studies of the role of Mn2+

As determined by EPR, malic enzyme from pigeon liver binds Mn2+ with a half-site stoichiometry of two tight binding sites (KD=6 to 10 mum) per enzyme tetramer and at two to four weak binding sites (KD=0.43 to 1.34 mM). The activation of malic enzyme by Mn2+ at high levels of L-malate shows biphasic kinetics yielding two activator constants for Mn2+. The dissociation constants of Mn2+ for both classes of sites are of the same order as the kinetically determined activator constants of Mn2+, indicating active site binding at both classes of binding sites. The binding of Mn2+ to the tight sites enhances the paramagnetic effect of Mn2+ on 1/T1 of water protons by a factor (epsilon) of 17, while binding at the weak sites yields a smaller epsilon of 11. The coenzymes TPN and TPNH have no effects on epsilon, while the carboxylic acid substrates L-malate and pyruvate and the inhibitors D-malate and oxalate significantly decrease epsilon. TPNH causes a 38-fold tightening of binding of the substrate L-malate to the enzyme-Mn2+ complex, consistent with the previously described highly ordered kinetic scheme, but only a 2-fold tightening of binding of the competitive inhibitor D-malate. The dissociation constant of L-malate from the quaternary E-Mn2+-TPNH-L-malate complex (32 muM) agrees with the Km of L-malate (25 muM), indicating active site binding. The dissociation constants of pyruvate from the ternary E-Mn2+-pyruvate complex (12 mM) and from the quaternary E-Mn2+-TPN-pyruvate complex (20 mM) are similar to the Km of pyruvate (5 mM), also indicating active site binding and a less highly ordered kinetic scheme for the reactions of pyruvate than for those of L-malate. Analysis of the frequency dependence of 1/T1 of water protons indicates that two fast exchanging water ligands remain coordinated to Mn2+ in the binary E-Mn2+ complex. The binding of the substrates L-malate and pyruvate and of the transition state analog oxalate to the E-Mn2+ complex decrease the number of fast exchanging water ligands on Mn2+ by approximately 1, but the binding of D-malate has no significant effect on this parameter, indicating the occlusion or replacement of a water ligand of the enzyme-bound Mn2+ by a properly oriented substituent on C-2 of the substrate. Occlusion rather than replacement of a water ligand by pyruvate is established by studies of 1/T1 of 13COO- and 13CO-enriched pyruvate which indicate second sphere Mn2+ to pyruvate distances of 4.6 A (COO-) and 4.8 A (CO) in the ternary enzyme-Mn2+-pyruvate complex. Formation of the quaternary complex with TPN increases these distances by 0.8 A, indicating the participation of a second sphere enzyme-Mn2+-(H2O)-pyruvate complex in catalysis. Thus, malic enzyme, like five other enzymes which utilize metals to polarize carbonyl groups, forms a second sphere complex with its substrate.     

The molybdoenzyme system of Drosophila melanogaster. I. Sulfite oxidase: identification and properties. Expression of the enzyme in maroon-like (mal), low-xanthine dehydrogenase (lxd), and cinnamon (cin) flies

Sulfite oxidase (sulfite: ferricytochrome c oxidoreductase; EC 1.8.2.1) has been detected in Drosophila melanogaster and some of its properties have been studied. In most respects this enzyme resembles the mammalian sulfite oxidases except for its molecular weight (148,000), which is somewhat higher than that of rat sulfite oxidase (116,000). Cytochrome c, potassium-ferricyanide, and oxygen can serve as electron acceptors in the oxidation of sulfite by the enzyme. Although definite evidence can be obtained only through the analysis of the pure enzyme, experiments involving tungstate feeding suggest that Drosophila sulfite oxidase is most probably a molybdoenzyme. Extracts of mal flies show normal levels of sulfite oxidase, whereas lxd flies have only 5-10% of the activity of wild type, and in cin flies the enzyme is apparently absent. While it is possible that the lxd and cin mutations are at some level responsible for the defective synthesis of a molybdenum-containing cofactor (supposed to be present in most molybdoenzymes), the evidence accumulated so far by several authors and the results of the present investigation argue against the involvement of a Mo cofactor in the multiple enzyme deficiencies observed in mal flies.     

Cytosolic isocitrate dehydrogenase in humans, mice, and voles and phylogenetic analysis of the enzyme family

In this study, we report cDNA sequences of the cytosolic NADP-dependent isocitrate dehydrogenase for humans, mice, and two species of voles (Microtus mexicanus and Microtus ochrogaster). Inferred amino acid sequences from these taxa display a high level of amino acid sequence conservation, comparable to that of myosin beta heavy chain, and share known structural features. A Caenorhabditis elegans enzyme that was previously identified as a protein similar to isocitrate dehydrogenase is most likely the NADP-dependent cytosolic isocitrate dehydrogenase enzyme equivalent, based on amino acid similarity to mammalian enzymes and phylogenetic analysis. We also suggest that NADP-dependent isocitrate dehydrogenases characterized from alfalfa, soybean, and eucalyptus are most likely cytosolic enzymes. The phylogenetic tree of various isocitrate dehydrogenases from eukaryotic sources revealed that independent gene duplications may have given rise to the cytosolic and mitochondrial forms of NADP-dependent isocitrate dehydrogenase in animals and fungi. There appears to be no statistical support for a hypothesis that the mitochondrial and cytosolic forms of the enzyme are orthologous in these groups. A possible scenario of the evolution of NADP-dependent isocitrate dehydrogenases is proposed.     

Construction and analysis of a semi-quantitative energy profile for the reaction catalyzed by the radical enzyme galactose oxidase

The investigation of the effect of oxidized lipoproteins on platelet activity is important for the understanding of the plague formation under atherosclerosis. In the present work, we examined the influence of low density lipoproteins (LDL) on ADP-induced platelet aggregation in the platelet rich plasma. In was demonstrated that mixing of plasma and LDL was accompanied by the decrease of ADP-induced aggregation parameters as compared to control (mixing with buffer). After 1 h incubation, platelet ADP-aggregation in the sample containing oxidized LDL (oxLDL) exceeded the ADP-aggregation in the control sample. The dependence of the aggregation parameters on the incubation time and on the degree of LDL oxidation were obtained. No difference in the cholesterol and phospholipid content was observed between cells incubated with buffer, native or oxidized LDL. Therefore, the possible oxLDL-induced accumulation of cholesterol in platelet membranes is excluded as a reason for the increased cell aggregation.     

A flavoprotein functional as NADH oxidase from Amphibacillus xylanus Ep01: purification and characterization of the enzyme and structural analysis of its gene

Amphibacillus xylanus Ep01, a facultative anaerobe we recently isolated, shows rapid aerobic growth even though it lacks a respiratory pathway. Thus, the oxidative consumption of NADH, produced during glycolysis and pyruvate oxidation, should be especially important for maintenance of intracellular redox balance in this bacterium. We purified a flavoprotein functional as NADH oxidase from aerobically growing A. xylanus Ep01. The A. xylanus enzyme is a homotetramer composed of a subunit (M(r) 56,000) containing 1 mol of flavin adenine dinucleotide. This enzyme catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH as the preferred electron donor and exhibits no activity with NADPH. The flavoprotein gene of A. xylanus Ep01 was cloned by using a specific antibody. The amino acid sequence of 509 residues, deduced from the nucleotide sequence, showed 51.2 and 72.5% identities to the amino acid sequences of alkyl hydroperoxide reductase from Salmonella typhimurium and NADH dehydrogenase from alkalophilic Bacillus sp. strain YN-1, respectively. Bacillus spp. have a respiratory chain and grow well under aerobic conditions. In contrast, Amphibacillus spp., having no respiratory chain, grow equally well under both aerobic and anaerobic conditions, which distinguishes these two genera. Salmonella spp., which are gram-negative bacteria, are taxonomically distant from gram-positive bacteria such as Bacillus spp. and Amphibacillus spp. The above findings, however, suggest that the flavoprotein functional as NADH oxidase, the alkyl hydroperoxide reductase, and the NADH dehydrogenase diverged recently, with only small changes leading to their functional differences.     

Sequence of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Nicotiana plumbaginifolia and phylogenetic origin of the gene family

A cDNA-library has been constructed from Nicotiana plumbaginifolia seedlings, and the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GapN, EC 1.2.1.9) was isolated by plaque hybridization using the cDNA from pea as a heterologous probe. The cDNA comprises the entire GapN coding region. A putative polyadenylation signal is identified. Phylogenetic analysis based on the deduced amino acid sequences revealed that the GapN gene family represents a separate ancient branch within the aldehyde dehydrogenase superfamily. It can be shown that the GapN gene family and other distinct branches of the superfamily have its phylogenetic origin before the separation of primary life-forms. This further demonstrates that already very early in evolution, a broad diversification of the aldehyde dehydrogenases led to the formation of the superfamily.     

Assembly and activation of the phagocyte NADPH oxidase. Specific interaction of the N-terminal Src homology 3 domain of p47phox with p22phox is required for activation of the NADPH oxidase

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (KD = 0.34 microM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.