Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.     

A novel MN2+-oxidizing enzyme system in a freshwater bacterium

Manganese oxidation by cell suspensions and cell extracts of a freshwater bacterium, designated strain FMn 1, was investigated. Manganese appeared to be oxidized in the periplasmic space. A conventional, membrane-bound-electron transport system was not utilized. An enzyme or enzyme complex and a cofactor, each of different molecular size, were located in different parts of the cell envelope. Results suggest that the cofactor reacts with manganese in the periplasmic space and that in the presence of oxygen it is reoxidized by the enzyme. The enzyme is probably loosely bound to the membrane. A combination of enzyme and cofactor in a crude preparation exhibited a pH optimum at around 7.0. The enzyme exhibited a temperature optimum at around 30 degrees C. No temperature optimum was found for the cofactor. The enzyme was heat-stable and could oxidize manganese under anaerobic conditions. The enzyme system appears to be different from others so far described.     

Kinetic and nuclear magnetic resonance study of the interaction of NADP+ and NADPH with chicken liver fatty acid synthase

The ionic strength dependence of the second-order rate constant for the association of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and chicken liver fatty acid synthase was determined. This rate constant is 7.2 X 10(7) M-1 s-1 at zero ionic strength and 25 degrees C; the effective charge at the cofactor binding sites is +0.8. The conformations of nicotinamide adenine dinucleotide phosphate (NADP+) and NADPH bound to the beta-ketoacyl and enoyl reductase sites were determined from transferred nuclear Overhauser effect measurements. Covalent modification of the enzyme with pyridoxal 5'-phosphate abolished cofactor binding at the enoyl reductase site; this permitted the cofactor conformations at the beta-ketoacyl and enoyl reductase sites to be distinguished. For NADP+ bound to the enzyme, the conformation of the nicotinamide-ribose bond is anti at the enoyl reductase site and syn at the beta-ketoacyl reductase site; the adenine-ribose bond is anti, and the sugar puckers are C3'-endo. Nicotinamide-adenine base stacking was not detected. Structural models of NADP+ at the beta-ketoacyl and enoyl reductase sites were constructed by using the distances calculated from the observed nuclear Overhauser effects. Because of the overlap of the resonances of several nonaromatic NADPH protons with the resonances of HDO and ribose protons, less extensive structural information was obtained for NADPH bound to the enzyme. However, the conformations of NADPH bound to the two reductases are qualitatively the same as those of NADP+, except that the nicotinamide moiety of NADPH is closer to being fully anti at the enoyl reductase site.     

Substrate and cofactor binding to fluorescently labeled cytoplasmic malate dehydrogenase

Cytoplasmic malate dehydrogenase (cMDH) is a key enzyme in several metabolic pathways. Though its activity has been examined extensively, there are lingering mechanistic uncertainties involving substrate and cofactor binding. To more completely understand this enzyme's interactions with cofactor and substrate ligands, a fluorescent reporter group was introduced into the enzyme's structure. This was accomplished by selective modification of Cys 110. The reaction placed an aminonaphthaline sulfonic acid group near the enzyme's active site. Substrate, inhibitor, and NAD binding activities were characterized using changes in this label's fluorescence. Results demonstrated that both substrate and cofactor molecules bound to the enzyme in the absence of their companion ligands. This is in contrast to strictly ordered cofactor then substrate binding as has been suggested by kinetic analyses of closely related enzymes. Binding results also indicated that the cofactor, NAD, bound to cMDH in a negatively cooperative manner, but substrates and the inhibitor, hydroxymalonate, bound non-cooperatively. Multiple substrate binding modes were identified and interactions between substrate and cofactor binding were found.     

Acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase. Evidence for an active site histidine residue

The lysosomal membrane enzyme acetyl-CoA:alpha-glucosaminide N-acetyltransferase catalyzes the transfer of the acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction appears to be a transmembrane process: the enzyme is acetylated on the outside of the lysosome, and the acetyl group is transferred across the membrane to the inside of the lysosome where it is used to acetylate glucosamine. To determine the reactive site residues involved in the acetylation reaction, lysosomal membranes were treated with various amino acid modification reagents and assayed for enzyme activity. Although four thiol modification reagents were examined, only one, p-chloromercuribenzoate inactivated the N-acetyltransferase. Thiol modification by p-chloromercuribenzoate did not appear to occur at the active site since inactivation was still observed in the presence of the substrate acetyl-CoA. N-Acetyltransferase could be inactivated by N-bromosuccinimide, even after pretreatment with reagents specific for tyrosine and tryptophan, suggesting that the modified residue is a histidine. Diethyl pyrocarbonate, another histidine modification reagent, could also inactivate the enzyme; this inactivation could be reversed by incubation with hydroxylamine. N-Bromosuccinimide and diethyl pyrocarbonate modifications appear to be at the active site of the enzyme since co-incubation with acetyl-CoA protects the N-acetyltransferase from inactivation. This protection is lost if glucosamine is also present. Pre-acetylated lysosomal membranes are also able to provide protection from N-bromosuccinimide inactivation, providing further evidence for a histidine moiety at the active site and for the existence of an acetyl-enzyme intermediate.     

Activation of nit-1 nitrate reductase by W-formate dehydrogenase

Formate dehydrogenase ( FDH ) from Clostridium thermoaceticum is a known tungsten enzyme. FDH was tested for the presence of nitrogenase-type cofactor and nitrate reductase-type cofactor by the Azotobacter vinelandii UW-45 and Neurospora crassa nit-1 reconstitution assays, respectively. Tungsten formate dehydrogenase (W- FDH ), containing only a small Mo impurity, activated the nit-1 nitrate reductase extracts when molybdate was also added, but not when tungstate was added. These results show W- FDH contains the cofactor common to all known Mo-enzymes except nitrogenase. The difference between the redox chemistries of W- FDH and W-substituted sulfite oxidase appears to relate to differences in tungsten ligation other than that donated by the cofactor or to variations in the protein environment surrounding the tungsten active site.     

Role of aspartate-37 in determining cofactor specificity and binding in rat liver dihydropteridine reductase

Full-length rat dihydropteridine reductase (DHPR) cDNAs have been combined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express dihydropteridine reductase immunoreactive proteins and demonstrate conversion of quinonoid dihydropteridines to their tetrahydro forms. Several recombinant enzymes have been purified to homogeneity and biochemical studies have been carried out comparing their properties with those exhibited by the rat liver enzyme. The optimal reaction conditions, kinetic constants, and stability are similar for the recombinant and naturally occurring enzyme. The results indicate that the nonmutant recombinant rat DHPR is an authentic replica of the natural protein and that the characteristics of DHPR activity are determined by a single gene product and do not require specific modification via the eukaryotic cell. In addition to the wild type, three specific mutagenic forms of the reductase, A-6-V, W-104-F, and D-37-I, and an additional abbreviated structure have also been formed. Each of the products exhibits reductase activity, although they show varied affinities for their cofactor, NADH, and less stability to chromatography, dialysis, and concentration than the wild-type enzyme. The N-terminal sequence contains a classic NADH binding region between amino acids 9 and 36, and Asp 37 is essential for binding the cofactor as is shown by the approximately 20-fold increase in dissociation constant for the D-37-I mutant and diminished kcat (approximately 43 s-1 compared to 156 s-1 for the wild-type enzyme). The results indicate that the DHPR cofactor binding site is similar to typical dinucleotide requiring dehydrogenases such as lactic acid and liver alcohol dehydrogenase.     

9-(5',6'-dideoxy-beta-D-ribo-hex-5'-ynofuranosyl)adenine, a novel irreversible inhibitor of S-adenosylhomocysteine hydrolase

The acetylenic analogue of adenosine 9-(5',6'-dideoxy-beta-D-ribo-hex-5'-ynofuranosyl)adenine has been synthesized, and its behavior as an inhibitor of bovine S-adenosylhomocysteine hydrolase has been examined. Incubation of the enzyme with excess inhibitor caused a time-dependent, irreversible inactivation of the enzyme that was accompanied by the reduction of two equivalents of NAD+ to NADH and the loss of the two remaining equivalents of NAD+. With use of radiolabeled inhibitor, it was established that 4 equiv of the acetylenic analog bind irreversibly to the enzyme and that 4 equiv were required to inactivate the enzyme completely. The inactivated enzyme could not be reactivated by incubation with NAD+. Denaturation studies revealed that 2 equiv of the inhibitor are bound more tightly to the enzyme than the remainder, suggesting the formation of a covalent linkage between the oxidized inhibitor and the enzyme. The putative covalent linkage was found to be acid sensitive but stable to mild base. The linkage could not be stabilized by treatment of the enzyme-inhibitor complex with either borohydride or cyanoborohydride. A Kl of 173 nM was measured for the inhibitor, making it one of the more potent inhibitors that have been reported. The enzyme used in these studies was isolated by modification of an affinity chromatography method reported by Narayanan and Borchardt [(1988) Biochim. Biophys. Acta 965, 22-28]. The affinity chromatography unexpectedly led to the isolation of two forms of the enzyme. The major form contained 4.0 mol of nucleotide cofactor/mol of enzyme tetramer, while the minor form carried only 2.0 mol/tetramer.     

Binding of ATP to Brain Glutamate Decarboxylase as Studied by Affinity Chromatography

Abstract: The interactions of two forms of porcine brain glutamate decarboxylase (β-GAD and γ-GAD) with the effector ATP were studied by affinity chromatography. A third form, γk-GAD, was only slightly retarded by the affinity matrix and was eluted in the buffer wash. The interaction of GAD with the ATP affinity matrix was qualitatively similar to its interaction with free ATP as reported in previous kinetic studies. The rank order of adenine nucleotides as eluting agents and affinity ligands was ATP > ADP > AMP. GAD was also eluted by its cofactor, pyridoxal 5'-phosphate, and this was enhanced by 1 mM P_i In contrast, a high concentration (140 mM) of P_i by itself was required to elute the enzyme. GAD remained active while bound to the affinity column and was eluted in the holoenzyme form by ATP, indicating that the affinity ligand did not bind in the active site and did not displace catalytically active cofactor from the enzyme.     

Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle

Escherichia coli dihydrofolate reductase (DHFR) has several flexible loops surrounding the active site that play a functional role in substrate and cofactor binding and in catalysis. We have used heteronuclear NMR methods to probe the loop conformations in solution in complexes of DHFR formed during the catalytic cycle. To facilitate the NMR analysis, the enzyme was labeled selectively with [(15)N]alanine. The 13 alanine resonances provide a fingerprint of the protein structure and report on the active site loop conformations and binding of substrate, product, and cofactor. Spectra were recorded for binary and ternary complexes of wild-type DHFR bound to the substrate dihydrofolate (DHF), the product tetrahydrofolate (THF), the pseudosubstrate folate, reduced and oxidized NADPH cofactor, and the inactive cofactor analogue 5,6-dihydroNADPH. The data show that DHFR exists in solution in two dominant conformational states, with the active site loops adopting conformations that closely approximate the occluded or closed conformations identified in earlier X-ray crystallographic analyses. A minor population of a third conformer of unknown structure was observed for the apoenzyme and for the disordered binary complex with 5,6-dihydroNADPH. The reactive Michaelis complex, with both DHF and NADPH bound to the enzyme, could not be studied directly but was modeled by the ternary folate:NADP(+) and dihydrofolate:NADP(+) complexes. From the NMR data, we are able to characterize the active site loop conformation and the occupancy of the substrate and cofactor binding sites in all intermediates formed in the extended catalytic cycle. In the dominant kinetic pathway under steady-state conditions, only the holoenzyme (the binary NADPH complex) and the Michaelis complex adopt the closed loop conformation, and all product complexes are occluded. The catalytic cycle thus involves obligatory conformational transitions between the closed and occluded states. Parallel studies on the catalytically impaired G121V mutant DHFR show that formation of the closed state, in which the nicotinamide ring of the cofactor is inserted into the active site, is energetically disfavored. The G121V mutation, at a position distant from the active site, interferes with coupled loop movements and appears to impair catalysis by destabilizing the closed Michaelis complex and introducing an extra step into the kinetic pathway.     

4-Aminobutyrate aminotransferase. Conformational changes induced by reduction of pyridoxal 5-phosphate

Conformational changes induced in 4-aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) by conversion of pyridoxal-5-P to pyridoxyl-5-P were examined by two independent methods. The reactivity of the SH groups of the reduced enzyme is increased by chemical modification of the cofactor. 1.8 SH per dimer of modified enzyme react with DTNB, whereas 1.2 SH per dimer of the native enzyme react with the attacking reagent under identical experimental conditions. The modified and native forms of the enzyme bind the fluorescent probe ANS, but the number of binding sites for ANS is increased as result of conversion of P-pyridoxal to P-pyridoxyl. After the conformational changes onset by reduction of the cofactor, the modified enzyme binds one molecule of pyridoxal-5-P with a Kd of 0.1 microM to become catalytically competent. The catalytic site of the reduce enzyme was probed with P-pyridoxal analogs. Like resolved 4-aminobutyrate aminotransferase, the reduced species recognize the phosphorothioate analog and regain 40% of the total enzymatic activity. Since the catalytic parameters of reduced and native 4-aminobutyrate aminotransferase are indistinguishable, it is concluded that the additional catalytic site of the reduced enzyme is functionally identical to that of the native enzyme.     

Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity.

1. Isoelectric focusing studies of human placental diamine oxidase showed the pI value of the active enzyme to be 6.5. This information was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH gradient elution and this, together with affinity chromatography on concanavalin A--Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration of diamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultracentrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx. 1.0 and 1.2g-atom respectively/70000mol.wt. unit. 5. The e.s.r. spectral intensity did not decrease nor did the spectral line shape change when excess of p-dimethylaminomethylbenzylamine was added to the enzyme. 6. Addition of K13CN to the enzyme eliminated the copper e.s.r. signal without affecting the manganese signal. 7. The placental enzyme therefore appears to differ from other amine oxidases in terms of its metal cofactor requirement, molecular weight and substrate specificity, and possible roles in vivo for this enzyme are discussed.     

Purification and properties of an extracellular collagenolytic protease produced by the human oral bacterium Bacillus cereus (strain Soc 67)

The major collagenolytic proteinase present in the culture filtrate of Bacillus cereus (strain Soc 67, isolated from the human oral cavity) has been purified to homogeneity by a procedure that comprised concentration of ultrafiltered growth medium on a Millipore PTTK00005 membrane, precipitation with ammonium sulfate, gel permeation chromatography, chromatofocusing, fast protein liquid chromatography on an anion-exchange column, and finally fast protein liquid chromatography on a gel column. The enzyme hydrolyzed, with decreasing rates, phenylazobenzyloxy-carbonyl-L-Pro-L-Leu Gly-L-Pro-D-Arg (PZ-PLGPA), furylacrylolyl-L-Leu-Gly-L-Pro-L-Ala, and furylacryloyl-L-Phe-Gly-Gly, while furylacryloyl-Gly-L-Leu-NH2 was not hydrolyzed. The enzyme degraded soluble and insoluble collagens, Azocoll and gelatin. Bradykinin was hydrolyzed at a high rate at the Phe-Ser bond. The enzyme was sensitive to pyrophosphate, L-cysteine, and L-histidine and could be totally inactivated in the presence of metal chelators. The enzyme contains 1 mol of Zn/mol and the hydrolysis of PZ-PLGPA is slightly increased by Ca2+. The enzyme is readily inhibited by heavy metal cations, but Cu2+ and Ni2+ affected the catalysis in opposite ways: increasing levels of Cu2+ decreased the affinity of the enzyme for PZ-PLGPA, whereas Ni2+ had no effect. The effect of Cu2+ also depended on the pH and type of buffer used. Detailed chemical modification experiments suggested that the active site of the enzyme contains at least 1 tyrosyl and 1 lysyl residue, and 1 carboxyl group. The enzyme was not sensitive to sulfhydryl reagents and thiols did not activate the enzyme. The modification studies were unable to reveal active histidyl residues. The ability of the enzyme to hydrolyze PZ-PLGPA, furylacryloyl-L-Leu-Gly-L-Pro-L-Ala, furylacryloyl-L-Phe-Gly-Gly, and various collagenous materials, its inactivity toward furylacryloyl-Gly-L-Leu-NH2, and the results from the chemical modification studies suggest that the B. cereus (Soc 67) collagenolytic enzyme can be regarded as a true collagenase which resembles the Clostridium histolyticum collagenase(s).     

3,4,5,6-Tetrahydrophthalic anhydride modification of glutamate dehydrogenase: the construction and activity of heterohexamers

Modification of glutamate dehydrogenase with 3,4,5,6-tetrahydrophthalic anhydride at pH 8.0 results in the progressive loss of enzymatic activity and a concomitant increase in the negative charge of the protein. Although the rate of inactivation at room temperature is too rapid to allow accurate rate constant determination, modification at 4 degrees C shows that the pseudo-first-order rate constant for inactivation appears to show a saturation effect with increasing reagent concentration, with a maximum of approximately 1 min-1. Control experiments showed that tetrahydrophthalic anhydride was hydrolyzed at a much slower rate, with a pseudo-first-order rate constant of 0.041 min-1. Protection studies indicated that inactivation was decreased by the active site ligands, NADP and 2-oxoglutarate. The extents of inactivation, whether assayed with glutamate at pH 7.0 or norvaline at pH 8.0, were the same. Changes in mobility on native gels and isoelectric point were used to follow the incorporated negative charge resulting from modification. Enzyme modified in the presence of protecting ligands (where activity is maintained) showed mobility changes which suggested that a single site of modification was protected. Modified enzyme incorporated 0.78 mol pyridoxal 5-phosphate less than native enzyme, consistent with modification of lysine-126. Enzyme modified under limiting conditions was shown to have a quaternary structure similar to that of the native enzyme, as judged by crosslinking patterns obtained with dimethylpimelimidate. The modified protein is readily resolved from unmodified protein using an NaCl double gradient elution from DEAE-Sephacel. The modification is reversed with regain of activity by incubation of the modified enzyme at low pH. We have made use of the recently demonstrated ability of guanidine hydrochloride to dissociate the hexamer of glutamate dehydrogenase into trimers that can then be reassociated to construct heterohexamers of glutamate dehydrogenase, in which one trimer of the heterohexamer contains native subunits while the other has been inactivated by the 3,4,5,6-tetrahydrophthalic anhydride modification. The heterohexamer is separated from either native or fully modified hexamers by DEAE-Sephacel chromatography. Significantly, the heterohexamer has little detectable catalytic activity, although activity is regained by reversal of the modification of the one modified trimer in the hexamer. This demonstrates that catalytic site cooperation between trimers in the hexamer of glutamate dehydrogenase is an essential component of the enzymatic activity of this enzyme.     

Isolation and sequence of the pyridoxal 5'-phosphate active-site peptide from Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was modified with pyridoxal 5'-phosphate and then reduced with sodium borohydride. Both carboxylase and oxygenase activities were lost when one molecule of pyridoxal 5'-phosphate was bound per enzyme dimer. Peptide maps of modified enzyme showed one N6-(phosphopyridoxal)lysine-containing peptide. This peptide was isolated by gel filtration and cation-exchange chromatography and its sequence determined as Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP-Lys)-Gly-Thr-Leu-Val-Ile-Lys. Since activation of the enzyme with Mg2+/CO2 enhances pyridoxal 5'-phosphate modification and subsequent inactivation and the substrate ribulose bisphosphate protects against modification, the modified lysyl group is most certainly at the catalytic site and not at the activation site of the enzyme.     

Purification and characterization of NADH oxidase from Serpulina (Treponema) hyodysenteriae.

NADH oxidase (EC 1.6.99.3) was purified from cell lysates of Serpulina (Treponema) hyodysenteriae B204 by differential ultracentrifugation, ammonium sulfate precipitation, and chromatography on anion-exchange, dye-ligand-affinity, and size-exclusion columns. Purified NADH oxidase had a specific activity 119-fold higher than that of cell lysates and migrated as a single band during denaturing gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]). The enzyme was a monomeric protein with an estimated molecular mass of 47 to 48 kDa, as determined by SDS-PAGE and size-exclusion chromatography. Optimum enzyme activity occurred in buffers with a pH between 5.5 and 7.0. In the presence of oxygen, beta-NADH but not alpha-NADH, alpha-NADPH, or beta-NADPH was rapidly oxidized by the enzyme (Km = 10 microM beta-NADH; Vmax = 110 mumol beta-NADH min-1 mg of protein-1). Oxygen was the only identified electron acceptor for the enzyme. On isoelectric focusing gels, the enzyme separated into three subforms, with isoelectric pH values of 5.25, 5.35, and 5.45. Purified NADH oxidase had a typical flavoprotein absorption spectrum, with peak absorbances at wavelengths of 274, 376, and 448 nm. Flavin adenine dinucleotide was identified as a cofactor and was noncovalently associated with the enzyme at a molar ratio of 1:1. Assays of the enzyme after various chemical treatments indicated that a flavin cofactor and a sulfhydryl group(s), but not a metal cofactor, were essential for activity. Hydrogen peroxide and superoxide were not yielded in significant amounts by the S. hyodysenteriae NADH oxidase, indirect evidence that the enzyme produces water from reduction of oxygen with NADH. The N-terminal amino acid sequence of the NADH oxidase was determined to be MKVIVIGCHGAGTWAAK. In its biochemical properties, the NADH oxidase of S. hyodysenteriae resembles the NADH oxidase of another intestinal bacterium, Enterococcus faecalis.     

Purification and characterization of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney

A NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) from porcine kidney was purified to homogeneity by acid precipitation, blue agarose affinity chromatography, hydroxyapatite-ultrogel adsorption chromatography, DEAE-Sephadex ion-exchange chromatography and NAD(+)-agarose affinity chromatography. The specific activity of the homogeneous enzyme was 31.2 U/mg. The molecular mass of the native enzyme was estimated to be 55,000 Da, whereas that of SDS-treated enzyme was 29,000 Da indicating that the native enzyme was dimeric. Compared to human placental 15-OH-PGDH, porcine kidney enzyme gave a similar general amino acid residue distribution. Chemical modification of the enzyme with N-ethyl maleimide (3 microM), N-chlorosuccinimide (20 microM) or 2,4,6-trinitrobenzenesulfonic acid (2.5 microM) followed pseudo-first-order inactivation kinetics, and inactivation could be prevented by the presence of NAD+ (1 mM) but not of prostaglandin E1 (140 microM) indicating the involvement of cysteine, methionine and lysine residues in the coenzyme binding site. Inactivation by diethyl pyrocarbonate (1.25 mM) or phenylglyoxal (10 mM) also showed pseudo-first-order kinetics suggesting that histidine and arginine residues were catalytically or structurally important in the native enzyme. These studies provide new insights into the structure and function of 15-OH-PGDH.     

The formaldehyde dehydrogenase of Rhodococcus erythropolis, a trimeric enzyme requiring a cofactor and active with alcohols

During growth on compounds containing methyl groups a formaldehyde dehydrogenase is induced in the gram-positive bacteria Rhodococcus erythropolis. This formaldehyde dehydrogenase has been purified to homogeneity using affinity chromatography and permeation chromatography. The isoelectric point of the enzyme was 4.7. The molar mass of the native enzyme was determined as 130 000 g/mol. Sodium dodecyl sulfate gel electrophoresis yielded a single subunit with a molar mass of 44000 g/mol. These results, together with cross-linking experiments which yielded monomer, dimer, and trimer bands, are consistent with a trimeric subunit structure of the formaldehyde dehydrogenase. A heat-stable cofactor of low molar mass was required for activity with formaldehyde as substrate. This cofactor was found to be oxidizable, but active only in its reduced form. Preparative electrofocusing revealed that the cofactor is a weak acid with a pK of about 6.5. The enzyme was active with the homologous series of the primary alcohols, ethanol up to octanol, without requiring the presence of the cofactor. A mutant without formaldehyde dehydrogenase activity was not impaired in its growth with ethanol as substrate. It is suggested that the alcohols mimic the true substrate of the formaldehyde dehydrogenase, which could be a hydroxymethyl derivative of the cofactor, resulting from the addition of formaldehyde.     

Modification of the F0 portion of the H+-translocating adenosinetriphosphatase complex of Escherichia coli by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and effect on the proton channeling function

1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), a water-soluble carbodiimide, inhibited ECF1-F0 ATPase activity and proton translocation through F0 when reacted with Escherichia coli membrane vesicles. The site of modification was found to be in subunit c of the F0 portion of the enzyme but did not involve Asp-61, the site labeled by the hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD). EDC was not covalently incorporated into subunit c in contrast to DCCD. Instead, EDC promoted a cross-link between the C-terminal carboxyl group (Ala-79) and a near-neighbor phosphatidylethanolamine as evidenced by fragmentation of subunit c with cyanogen bromide followed by high-pressure liquid chromatography and thin-layer chromatography.     

Photodynamic properties of pyridoxal phosphate bound to cystathionase-gamma-lyase

The cofactor pyridoxal phosphate bound through an aldimine linkage to lysine residues of the enzyme cystathionase (L-Cystathione cysteine-lyase (deaminating), EC 4.4.1.1) is very stable to irradiation with light of 420 nm. The catalytic function of the enzyme remains unaffected indicating that the cofactor is not an efficient photosensitizer of essential amino acid residues. This unusual stability of the cofactor to irradiation can be ascribed to the presence of aldimine linkages as demonstrated by studies conducted on model compounds. The binding of a reversible inhibitor (L-allylglycine) to the catalytic site of the enzyme does not facilitate photooxidation of the cofactor. On the contrary, irradiation of the cofactor in the presence of the inhibitor results in photodestruction of the inhibitor.