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L-methionine degradation potentialities of cheese-ripening microorganisms.

Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase. The catabolism of L-methionine in these microorganisms is discussed.

Arthrobacter↗

Isomerization of trans-2,delta 5-dienoyl-CoA's to delta 3,delta 5-dienoyl-CoA's in the beta-oxidation of delta 5-unsaturated fatty acids.

The NADPH-dependent reduction pathway for the metabolism of delta 5-unsaturated fatty acids involves the isomerization of trans-2,delta 5-dienoyl-CoA, initially formed from the dehydrogenation of delta 5-enoyl-CoA, to isomeric delta 3,delta 5-dienoyl-CoA. The latter intermediates were then isomerized to trans-2,trans-4-dienoyl-CoA, which then follows the NADPH-dependent pathway mediated by 2,4-dienoyl-CoA reductase. The isomerization from trans-2,delta 5-dienoyl-CoA to delta 3,delta 5-dienoyl-CoA is catalyzed by delta 3,delta 2-enoyl-CoA isomerase. In this investigation, we identified the stereoisomers of delta 3,delta 5-dienoates that were formed in the reaction. Starting from trans-2,cis-5-decadienoyl-CoA, the isomerization produced cis-3,cis-5- and trans-3,cis-5-decadienoates. On the other hand, trans-2,trans-5-decadienoyl-CoA yielded cis-3,trans-5- and trans-3,trans-5-decadienoates. In addition to purified rat liver delta 3,delta 2-enoyl-CoA isomerase, acyl-CoA oxidase from Arthrobacter also catalyzed the isomerization from trans-2,cis-5-dienoyl-CoA. However, this acyl-CoA oxidase could not catalyze the similar isomerization of trans-2,trans-5-dienoyl-CoA. delta 3,delta 5-t-2,t-4-Dienoyl-CoA isomerase used cis-3,cis-5-, trans-3,cis-5-, and cis-3,trans-5-dienoyl-CoA's as substrates and converted them to trans-2,trans-4-dienoyl-CoA. In contrast, trans-3,trans-5-dienoyl-CoA was not a substrate for this isomerization. Extensive purification of acyl-CoA oxidase through column chromatography could not remove or diminish the isomerization activity associated with acyl-CoA oxidase. Acyl-CoA oxidases derived from Candida and rat liver also possess isomerization activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗

Spectroscopic studies on the mechanism of the topa quinone generation in bacterial monoamine oxidase.

Electron paramagnetic resonance (EPR), circular dichroism (CD), and optical absorption spectroscopies have been used to investigate the copper-dependent autoxidation process generating the 6-hydroxydopa (topa) quinone cofactor in the recombinant phenethylamine oxidase from Arthrobacter globiformis. The cupric ion bound to the copper/topa quinone-less, inactive enzyme is first reduced to Cu(I), as inferred from the spectroscopic features observed under strictly anaerobic conditions. Cu(I) is also detectable chemically with a Cu(I)-specific chelating agent, bathocuproinedisulfonate. Introduction of a limited amount of oxygen then leads to the formation of a paramagnetic species (g = 2.004) that is stable for over several to 10 min but vanishes swiftly upon addition of sufficient oxygen. Strikingly, the hyperfine EPR structure of the organic radical is almost identical with that of the topa semiquinolamine observed in the copper/topa quinone-containing, active enzyme anaerobically reduced with substrate. Concomitant with the generation of topa quinone exhibiting characteristic optical absorption and CD bands under fully aerobic conditions, the bound copper finally shows EPR signals typical of nonblue type II Cu(II) and optical absorption around 700 nm with negative CD above 700 nm. None of these spectral changes are evoked in the binding of Cu(II) to the Tyr382-->Phe mutant enzyme, indicating that the precursor Tyr382 to topa quinone participates in the initial reduction of bound copper and serves as the origin of the transiently formed semiquinone radical. The prosthetic cupric ion plays an essential role, by changing its redox state, in the oxidative modification of the tyrosyl phenol ring, leading to topa quinone.

Arthrobacter↗

Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-CoA and 12-[(4-azidosalicyl)amino]dodecanoyl-CoA.

Synthesis of 32P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [gamma-32P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture of oxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate. Acyl-CoA oxidase was incubated in the presence of acyl-CoA analogs and immediately photolyzed, which resulted in irreversible inhibition. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme from photoactivated inhibition by 12-azidooleoyl-CoA and ASD-CoA, respectively. Analysis of photolyzed enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that both analogs preferentially labeled a 54,000 molecular weight protein. These results demonstrate that the photoaffinity acyl-CoA analogs have potential application as probes to identify and characterize lipid biosynthetic enzymes and to identify the active site of these proteins.

Acyl Coenzyme A↗

Factors determining steric course of enzymic glycosylation reactions: glycosyl transfer products formed from 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol by alpha-glucosidases and an inverting exo-alpha-glucanase.

Glycosyl transfer products were formed from 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) by purified alpha-glucosidases from Candida tropicalis and rice and by an inverting exo-alpha-glucanase (glucodextranase) from Arthrobacter globiformis. The products were structurally defined through 1H and 13C NMR (nuclear magnetic resonance) spectra of their crystalline per-O-acetates in comparison with those of authentic methyl 1-deoxy-alpha- and methyl 1-deoxy-beta-D-gluco-heptuloside. 1-Deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-heptenitol and 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-D-gluco-heptulose were produced by both the Candida alpha-glucosidase and the glucodextranase; 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 5)- and 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-D-gluco-heptuloses by the rice alpha-glucosidase. These results, together with our earlier findings of sterospecific hydration of heptenitol catalyzed by the same enzymes [Hehre, E. J., Brewer, C. F., Uchiyama, T., Schlesselmann, P., & Lehmann, J. (1980) Biochemistry 19, 3557-3564], show the inadequacy of the long-accepted notion that carbohydrase-catalyzed reactions always lead to retention (or always lead to inversion) of substrate configuration. In particular, the finding that glucodextranase forms transfer products of alpha configuration and a hydration product of beta configuration from the same substrate provides a clear example of the functioning of acceptors rather than donor substrates in selecting the steric course of reactions catalyzed by a glycosylase. The circumstances under which acceptor cosubstrates might be expected to show this significant effect are discussed. The opportunity presumably would exist whenever carbonium ion mediated reactions are catalyzed by glycosylases that provide oppositely oriented approaches of different acceptors to the catalytic center.

Arthrobacter↗

Fish egg polysialoglycoproteins: circular dichroism and proton nuclear magnetic resonance studies of novel oligosaccharide units containing one sialidase-resistant N-glycolylneuraminic acid residue in each molecule.

Long-core units having the common sequence Ga1NAc beta 1 leads to 4(NeuGc2 leads to 3)Ga1NAc beta 1 leads to 3Gal beta 1 leads to 4Ga1 beta 1 leads to 3Ga1NAc are one of the major constituents of rainbow trout egg polysialoglycoproteins. The existing ambiguity regarding the anomeric configuration of the sialidase-resistant unsubstituted sialyl group present in this novel type of oligosaccharide chains has been resolved by a circular dichroism difference spectral method. The fact that the negative band originating from the carbohydrate n leads to pi transition for this sialyl group was observed offers conclusive proof of the alpha-anomeric configuration. Next particularly interesting is the fact that the chemical shifts of the sialidase-resistant sialyl H-3eq and H-3ax protons were respectively found at relatively higher and lower magnetic field than for the corresponding protons of other sialyl groups. A consideration of molecular models shows that the observed anomalies are all in the directions compatible with expectations on the basis of the magnetic anisotropy effect due to the carboxylate group and steric compression effects by van der Walls interactions between groups that are sterically compressed. In addition to the observed resistance to bacterial sialidases of this sialyl group, it did not behave even as a competitive inhibitor of the sialidase, Arthrobacter ureafaciens, indicating that inaccessibility of this unique sialyl group toward the enzyme. Finally, the analysis of the proton nuclear magnetic resonances of sialidase-sensitive mono- and oligosialyl groups present in the long-core units was based on comparisons of diagnostically important regions in the spectra of homologous oligosaccharides of N-glycolylneuraminic acid.

Animals↗

Stereochemical studies of D-glucal hydration by alpha-glucosidases and exo-alpha-glucanases: indications of plastic and conserved phases in catalysis by glycosylases.

Alpha-Glucosidases from Aspergillus niger, pig serum, ungerminated rice, buckwheat, and sugar beet seeds (but not from brewers' yeast or honeybee) were found to catalyze the hydration of D-glucal. Each reactive alpha-glucosidase, incubated with D-glucal in D2O, was shown to protonate (deuteriate) this prochiral substrate from above its re face, i.e., from a direction opposite that assumed for protonating alpha-D-glucosidic substrates. At the same time, D-glucal hydration catalyzed by three of the alpha-glucosidases that acted rapidly enough in D2O to determine product configuration was found to yield 2-deoxy-D-glucose of the same specific (alpha-) configuration as the D-glucose produced from alpha-D-glucosidic substrates. These findings substantially extend those reported earlier for the hydration of D-glucal by one (Candida tropicalis) alpha-glucosidase preparation. Together with other recent results, they suggest that the process of catalysis by alpha-glucosidases (and perhaps glycosylases in general) may comprise two separate and separately controlled parts, namely, a "plastic" phase concerned with substrate protonation and a substrate-unrelated "conserved" phase concerned with the creation of product configuration. In contrast to the alpha-glucosidases, three "inverting" exo-alpha-glucanases (Arthrobacter globiformis glucodextranase; Rhizopus niveus and Paecilomyces varioti glucoamylase) were found to protonate D-glucal from below its si face. Further, whereas the catalysis of D-glucal hydration by the alpha-glucosidases was intensively inhibited by excess substrate, that promoted by the exo-glucanases showed no detectable substrate inhibition.

Animals↗

Production and isolation of siderophores from the soil fungus Epicoccum purpurascens.

A large number of iron transport agents, siderophores, which stimulated the growth of Arthrobacter flavescens JG-9, were isolated during a study of the antitumor activity associated with the metabolic products of the fungus Epicoccum purpurascens. The production of the siderophores was significantly enhanced in a variety of media by culture of the fungus in the near absence of ferric iron. A novel method of purification involving a carboxylic ion-exchange resin separated the siderophores into four subgroups. The first subgroup, which contained the majority of the activity, was subsequently resolved in a similar manner with the carboxylic resin into seven individual siderophores. Of these, two were characterized as ferricrocin and coprogen whereas the others appeared to represent new compounds. One of the latter was given the name triornicin and exhibited slight antitumor activity in mice injected with Ehrlich ascites tumor cells.

Arthrobacter↗

The structure of L-hydantoinase from Arthobacter aurescens leads to an understanding of dihydropyrimidinase substrate and enantio specificity.

L-Hydantoinase from Arthrobacter aurescens (L-Hyd) is a member of the dihydropyrimidinases which in turn belong to the cyclic amidases. Dihydropyrimidinases catalyze the reversible hydrolytic ring opening of dihydropyrimidines as the second step in the catabolism of pyrimidines. In biotechnology, their hydroloytic activity on five-membered cyclic diamides (hydantoins) is used in the enantio-specific production of amino acids from racemic hydantoins. L-Hyd differs from most of the other dihydropyrimidinases by an L-enantio specificity and by lacking activity on possible natural substrates such as dihydropyrimidines. In this paper, we describe the three-dimensional structure of L-Hyd which was solved by molecular replacement using a homology model and subsequently refined to 2.6 A resolution. Each subunit of the tetrameric L-Hyd consists of an elliptically distorted (alpha/beta)(8)-barrel domain, which hosts the active site, and a beta-sheet domain. In the active site, a binuclear zinc center activates a water molecule for nucleophilic attack on the substrates' amide bond. L-Hyd shows a strong homology both in fold and in metal coordination in the active site to another dihydropyrimidinase from Thermus sp. (D-hydantoinase) and to a slightly lesser degree to ureases, dihydroorotase and phosphotriesterase. Using the homology to ureases, a model for the transition state was modeled in the active site of L-Hyd and D-hydantoinase. This model could provide an explanation for the different substrate and enantio selectivities of both dihydropyrimidinases.

Amidohydrolases↗

Chemical rescue of a site-specific mutant of bacterial copper amine oxidase for generation of the topa quinone cofactor.

The topa quinone (TPQ) cofactor of copper amine oxidase is produced by posttranslational modification of a specific tyrosine residue through the copper-dependent, self-catalytic process. We have site-specifically mutated three histidine residues (His431, His433, and His592) involved in binding of the copper ion in the recombinant phenylethylamine oxidase from Arthrobacter globiformis. The mutant enzymes, in which each histidine was replaced by alanine, were purified in the Cu/TPQ-free precursor form and analyzed for their Cu-binding and TPQ-generating activities by UV-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. Among the three histidine-to-alanine mutants, only H592A was found to show a weak activity to form TPQ upon aerobic incubation with Cu(2+) ions. Also for H592A, exogenous imidazole rescued binding of copper and markedly promoted the TPQ formation. Accommodation of a free imidazole molecule within the cavity created in the active site of H592A was suggested by X-ray crystallography. Although the TPQ cofactor in H592A mutant was readily reduced with substrate, its catalytic activity was very low even in the presence of imidazole. Combined with the crystal structures of the mutant enzymes, these results demonstrate the importance of the three copper-binding histidine residues for both TPQ biogenesis and catalytic activity, fine-tuning the position of the essential metal.

Alanine↗

Differential inhibition of six copper amine oxidases by a family of 4-(aryloxy)-2-butynamines: evidence for a new mode of inactivation.

A series of compounds derived from a previously identified substrate analogue of copper amine oxidases (CuAOs) (Shepard et al. (2002) Eur. J. Biochem. 269, 3645-3658) has been screened against six different CuAOs with a view to designing potent and selective inhibitors. The substrate analogues investigated were 4-(1-naphthyloxy)-2-butyn-1-amine, 4-(2-methylphenoxy)-2-butyn-1-amine, 4-(3-methylphenoxy)-2-butyn-1-amine, 4-(4-methylphenoxy)-2-butyn-1-amine, and 4-phenoxy-2-butyn-1-amine. These compounds were screened against equine plasma amine oxidase (EPAO), Pisum sativum amine oxidase (PSAO), Pichia pastoris lysyl oxidase (PPLO), bovine plasma amine oxidase (BPAO), human kidney diamine oxidase (KDAO), and Arthrobacter globiformis amine oxidase (AGAO) to examine the effect of different substituent groups on potency. Despite the similar structures of the 4-aryloxy analogues evaluated, striking differences in potency were observed. In addition, crystal structures of AGAO derivitized with 4-(2-naphthyloxy)-2-butyn-1-amine and 4-(4-methylphenoxy)-2-butyn-1-amine were obtained at a resolution of 1.7 A. The structures reveal a novel and unprecedented reaction mechanism involving covalent attachment of the alpha,beta-unsaturated aldehyde turnover product to the amino group of the reduced 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. Collectively, the structural and inhibition results support the feasibility of designing selective mechanism-based inhibitors of copper amine oxidases.

Allylamine↗

Reinvestigation of metal ion specificity for quinone cofactor biogenesis in bacterial copper amine oxidase.

The topa quinone (TPQ) cofactor of copper amine oxidase is generated by copper-assisted self-processing of the precursor protein. Metal ion specificity for TPQ biogenesis has been reinvestigated with the recombinant phenylethylamine oxidase from Arthrobacter globiformis. Besides Cu2+ ion, some divalent metal ions such as Co2+, Ni2+, and Zn2+ were also bound to the metal site of the apoenzyme so tightly that they were not replaced by excess Cu2+ ions added subsequently. Although these noncupric metal ions could not initiate TPQ formation under the atmospheric conditions, we observed slow spectral changes in the enzyme bound with Co2+ or Ni2+ ion under the dioxygen-saturating conditions. Resonance Raman spectroscopy and titration with phenylhydrazine provided unambiguous evidence for TPQ formation by Co2+ and Ni2+ ions. Steady-state kinetic analysis showed that the enzymes activated by Co2+ and Ni2+ ions were indistinguishable from the corresponding metal-substituted enzymes prepared from the native copper enzyme (Kishishita, S., Okajima, T., Kim, M., Yamaguchi, H., Hirota, S., Suzuki, S., Kuroda, S., Tanizawa, K., and Mure, M. (2003) J. Am. Chem. Soc. 125, 1041-1055). X-ray crystallographic analysis has also revealed structural identity of the active sites of Co- and Ni-activated enzymes with Cu-enzyme. Thus Cu2+ ion is not the sole metal ion assisting TPQ formation. Co2+ and Ni2+ ions are also capable of forming TPQ, though much less efficiently than Cu2+.

Amine Oxidase (Copper-Containing)↗

Kinetic and structural studies on the catalytic role of the aspartic acid residue conserved in copper amine oxidase.

Copper amine oxidase contains a post-translationally generated quinone cofactor, topa quinone (TPQ), which mediates electron transfer from the amine substrate to molecular oxygen. The overall catalytic reaction is divided into the former reductive and the latter oxidative half-reactions based on the redox state of TPQ. In the reductive half-reaction, substrate amine reacts with the C5 carbonyl group of the oxidized TPQ, forming the substrate Schiff base (TPQ(ssb)), which is then converted to the product Schiff base (TPQ(psb)). During this step, an invariant Asp residue with an elevated pKa is presumed to serve as a general base accepting the alpha proton of the substrate. When Asp298, the putative active-site base in the recombinant enzyme from Arthrobacter globiformis, was mutated into Ala, the catalytic efficiency dropped to a level of about 10(6) orders of magnitude smaller than the wild-type (WT) enzyme, consistent with the essentiality of Asp298. Global analysis of the slow UV/vis spectral changes observed during the reductive half-reaction of the D298A mutant with 2-phenylethylamine provided apparent rate constants for the formation and decay of TPQ(ssb) (k(obs) = 4.7 and 4.8 x 10(-4) s(-1), respectively), both of which are markedly smaller than those of the WT enzyme determined by rapid-scan stopped-flow analysis (k(obs) = 699 and 411 s(-1), respectively). Thus, Asp298 plays important roles not only in the alpha-proton abstraction from TPQ(ssb) but also in other steps in the reductive half-reaction. X-ray diffraction analyses of D298A crystals soaked with the substrate for 1 h and 1 week revealed the structures of TPQ(ssb) and TPQ(psb), respectively, as pre-assigned by single-crystal microspectrophotometry. Consistent with the stereospecificity of alpha-proton abstraction, the pro-S alpha-proton of TPQ(ssb) to be abstracted is positioned nearly perpendicularly to the plane formed by the Schiff-base imine double bond conjugating with the quinone ring of TPQ, so that the orbitals of sigma and pi electrons maximally overlap in the conjugate system. More intriguingly, the pro-S alpha proton of the substrate is released stereospecifically even in the reaction catalyzed by the base-lacking D298A mutant. On the basis of these results, we propose that the stereospecificity of alpha-proton abstraction is primarily determined by the conformation of TPQ(ssb), rather than the relative geometry of TPQ and the catalytic base.

Amine Oxidase (Copper-Containing)↗

A comparative study of the binding and inhibition of four copper-containing amine oxidases by azide: implications for the role of copper during the oxidative half-reaction.

Copper amine oxidases (CuAOs) catalyze the oxidative deamination of primary amines operating through a ping-pong bi-bi mechanism. In this work, azide (an exogenous monodentate ligand) was used to probe the role of copper during the oxidative half-reaction of CuAO catalysis. The effects of azide on both the reductive and oxidative half-reactions of pea seedling amine oxidase (PSAO), the recombinant human kidney diamine oxidase (rhDAO), Arthrobacter globiformis amine oxidase (AGAO), and Pichia pastoris amine oxidase (PPLO) have been examined. For the reductive half-reaction, defined as the oxidation of amine substrate to an aldehyde, azide was discovered to exhibit either noncompetitive or competitive inhibition with respect to the amine, depending on the enzyme source. With regard to the oxidative half-reaction, defined as the reoxidation of the enzyme via reduction of O(2) to H(2)O(2), azide has been determined to exhibit competitive inhibition with respect to O(2) in PSAO with a calculated K(i) value that is in excellent agreement with the experimentally determined K(d) value for the Cu(II)-N(3)(-) complex. Azide was found to exhibit mixed-type/partially competitive inhibition with respect to substrate O(2) in rhDAO, with an apparent K(i) that is similar to the K(d) value for the Cu(II)-N(3)(-) complex. The competitive inhibition for PSAO and the partially competitive inhibition for rhDAO are consistent with O(2) interacting directly with copper during enzymatic reoxidation. For the enzymes AGAO and PPLO, pure noncompetitive and mixed-type/partially competitive inhibition is observed. K(i) values for reductive and oxidative half-reactions are equivalent and are lower than measured K(d) values for the Cu(II)-N(3)(-) complexes in oxidized and substrate-reduced forms of these enzymes. Given these observations, it appears that substantial inhibition of the reductive half-reaction occurs at the concentrations of azide used for the oxidative half-reaction experiments, thereby complicating kinetic interpretation. At this time, the data do not permit us to distinguish between two possibilities: (1) inhibition by azide with respect to O(2) is intrinsically competitive in CuAOs, but this effect cannot always be deconvolved experimentally from the effects of azide on the reductive half-reaction; or (2) CuAOs differ in some steps of their reoxidation mechanisms.

Amine Oxidase (Copper-Containing)↗

Mechanistic studies of topa quinone biogenesis in phenylethylamine oxidase.

An alternative purification for apophenylethylamine oxidase from Arthrobacter globiformis has been developed, which avoids the use of possible contaminants that may interfere with the topa quinone (TPQ) self-processing reaction. The binding of Cu(II) and the kinetics of TPQ formation in these enzyme preparations have been reinvestigated. Our results show that Cu(II) is not significantly reduced when added to the apoprotein under anaerobic conditions. The Cu(II) EPR and circular dichroism spectra of the initially formed complex are different from the spectra of the mature Cu(II)/TPQ-containing protein, indicating that the active site structure must be altered during TPQ formation. The kinetics we observe are cleanly first-order in protein [measured subsequent to Cu(II) binding] when dioxygen is present in pseudo-first-order excess (k(obs) = 1.5 min(-1)). We found no rate dependence on copper, so long as one copper per subunit was present. This indicates that tyrosine oxidation to give TPQ depends only on the copper that is bound in the active site. These results differ from those originally reported; an alternative mechanism, which involves attack of an activated copper-oxygen species on a tyrosine radical intermediate, is proposed for TPQ formation.

Amine Oxidase (Copper-Containing)↗

Topaquinone-dependent amine oxidases: identification of reaction intermediates by Raman spectroscopy.

Resonance Raman (RR) spectroscopy has proven to be an excellent technique for providing structural information about the 2,4, 5-trihydroxyphenylalaninequinone (TPQ) cofactor and for identifying the source of oxygen atoms during the posttranslational synthesis of the cofactor. Through specific labeling of the C2, C4, and C5 oxygens of TPQ in phenylethylamine oxidase (PEAO) from Arthrobacter globiformis, we have identified the C=O stretch of the C5 carbonyl at 1683 cm-1 (-27 in 18O) and the C=O stretch of the C2 carbonyl at 1575 cm-1 (-21 in 18O). These vibrational frequencies show that the C-O moiety at C5 has far greater double-bond character than at C2 or C4, thereby explaining the exclusive nucleophilic attack at the C5 position by substrates and substrate analogs. Bovine serum amine oxidase (BSAO) exhibits a similar nu(C=O) mode at 1678 cm-1 (-22 cm-1 in 18O). Aniline reacts with the TPQ cofactor of PEAO to form a new derivative (lambdamax at 450 nm) with properties similar to the proposed substrate-imine intermediate in the catalytic cycle. It retains the C2=O spectral features of the native enzyme and exhibits a new C5=N stretch at 1603 cm-1 (-29 in 15N). In contrast, methylamine reacts with both PEAO and BSAO under anaerobic conditions to form a different stable adduct (lambdamax at 385 nm) with properties closer to the proposed product-imine intermediate in the catalytic cycle. This species has a distinctive RR spectrum with a C=N stretch at 1617 cm-1 that corresponds to the atoms of the added methylamine (-58 cm-1 with CD3NH2, -19 cm-1 with CH315NH2). The lack of D2O dependence of nu(C=N) shows that this is a deprotonated imine, which would be more stable toward hydrolysis than the postulated protonated imine in the enzymatic reaction. However, the BSAO product imine (from methylamine) does undergo hydrolysis and conversion to semiquinone upon addition of cyanide. It is possible that the inactive form of the product imine is stabilized by deprotonation and flipping of the TPQ ring [Cai, D., Dove, J., Nakamura, N., Sanders-Loehr, J., and Klinman, J. P. (1997) Biochemistry 36, 11472-11478].

Amine Oxidase (Copper-Containing)↗

Stoichiometry of the topa quinone biogenesis reaction in copper amine oxidases.

The stoichiometry of the topa quinone biogenesis reaction in phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been determined. We have shown that the 6e- oxidation of tyrosine to topa quinone (TPQ) consumes 2 mol of O2 and produces 1 mol of H2O2/mol of TPQ formed. The rate of H2O2 production is first-order (kobs = 1.0 +/- 0.2 min-1), a rate only slightly lower than the rate of TPQ formation directly determined previously (kobs = 1.5 +/- 0.2 min-1). This gives the following net reaction stoichiometry for TPQ biogenesis: E-Tyr + 2O2 --> E-TPQ + H2O2. This stoichiometry is in agreement with recently proposed mechanisms for TPQ biogenesis, and rules out several possible alternatives.

Amine Oxidase (Copper-Containing)↗

Electron spin resonance study of chromium(V) formation and decomposition by basalt-inhabiting bacteria.

Bacterial reduction of Cr(VI) to Cr(III) compounds may produce reactive intermediates Cr(V) and Cr(IV), which can affect the mobility and toxicity of chromium in environments. To address this important subject, we conducted an electron spin resonance (ESR) study to understand the kinetics of the formation and decomposition of Cr(V) during Cr(VI) reduction by different gram-positive Cr(VI)-tolerant bacteria, which were isolated from polluted basalts from the United States of America and the Republic of Georgia. Results from our batch experiments show that during Cr(VI) reduction, the macromolecules at the cell wall of these bacteria could act as an electron donor to Cr(VI) to form a stable square-pyramidal Cr(V) complexes, which were reduced further probably via a one-electron transfer pathway to form Cr(IV) and Cr(III) compounds. The Cr(V) peak at the ESR spectrum possessed superhyperfine splitting characteristic of the Cr(V) complexes with diol-containing molecules. It appears that the kinetics of Cr(V) formation and decomposition depended on the bacterial growth phase and on the species. Both formation and decomposition of Cr(V) occurred more quickly when Cr(VI) was added at the exponential phase. In comparison with other gram-positive bacteria from the republic of Georgia, the formation and decomposition of Cr(V) in Arthrobacter species from the Unites States was significantly slower.

Arthrobacter↗