Transient kinetics of copper-containing lentil (Lens culinaris) seedling amine oxidase.
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ABSTRACT:
SUBMITTER: Bellelli A
PROVIDER: S-EPMC1152971 | biostudies-other | 1985 Dec
REPOSITORIES: biostudies-other
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Project description:Spectrophotometry and rapid-scanning stopped-flow spectroscopy have been used to investigate the visible absorbance changes that occur in the course of the reduction of lentil (Lens esculenta) seedling amine oxidase by substrate. The catalytic cycle of the enzyme employs several intermediates but, owing to kinetic limitations, some of them were not identified in previous studies. In this study we have examined several substrates, either rapidly reacting (e.g. putrescine) or slowly reacting (e.g. gamma-aminobutanoic acid). Two forms of the enzyme, namely the Cu(I)-aminoresorcinol and quinone ketimine derivatives, whose characterization was elusive in previous studies, have been identified and assigned an optical spectrum. Moreover the reduced form of the enzyme is shown to be an equilibrium mixture of two species, the Cu(I)-semiquinolamine radical and Cu(II)-aminoresorcinol; these have been resolved by pH dependence and assigned spectra as well as a second-order rate constant for the reaction with oxygen. Thus the results presented here identify all the catalytic intermediates suggested by the chemical nature of the coenzyme and define their spectroscopic and reactivity properties.
| S-EPMC1219498 | biostudies-other
Project description:The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels. The necessity of these channels in the case of small substrates is questionable because diffusion through the protein matrix is often assumed. Copper amine oxidases contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide, respectively. The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha is explored using a combination of kinetic, x-ray crystallographic, and computational approaches. A crystal structure of H. polymorpha amine oxidase in complex with xenon gas, which serves as an experimental probe for molecular oxygen binding sites, reveals buried regions of the enzyme suitable for transient molecular oxygen occupation. Calculated O(2) free energy maps using copper amine oxidase crystal structures in the absence of xenon correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O(2) migration routes of differing probabilities within the protein matrix. Site-directed mutagenesis designed to block individual routes has little effect on overall k(cat)/K(m) (O(2)), supporting multiple dynamic pathways for molecular oxygen to reach the active site.
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Project description:Zinc-α2-glycoprotein (ZAG) is a major plasma protein whose levels increase in chronic energy-demanding diseases and thus serves as an important clinical biomarker in the diagnosis and prognosis of the development of cachexia. Current knowledge suggests that ZAG mediates progressive weight loss through β-adrenergic signalling in adipocytes, resulting in the activation of lipolysis and fat mobilization. Here, through cross-linking experiments, amine oxidase copper-containing 3 (AOC3) is identified as a novel ZAG binding partner. AOC3-also known as vascular adhesion protein 1 (VAP-1) and semicarbazide sensitive amine oxidase (SSAO)-deaminates primary amines, thereby generating the corresponding aldehyde, H2O2 and NH3. It is an ectoenzyme largely expressed by adipocytes and induced in endothelial cells during inflammation. Extravasation of immune cells depends on amine oxidase activity and AOC3-derived H2O2 has an insulinogenic effect. The observations described here suggest that ZAG acts as an allosteric inhibitor of AOC3 and interferes with the associated pro-inflammatory and anti-lipolytic functions. Thus, inhibition of the deamination of lipolytic hormone octopamine by AOC3 represents a novel mechanism by which ZAG might stimulate lipolysis. Furthermore, experiments involving overexpression of recombinant ZAG reveal that its glycosylation is co-regulated by oxygen availability and that the pattern of glycosylation affects its inhibitory potential. The newly identified protein interaction between AOC3 and ZAG highlights a previously unknown functional relationship, which may be relevant to inflammation, energy metabolism and the development of cachexia.
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Project description:BackgroundDrought stress is one of the most harmful abiotic stresses in crop plants. As a moderately drought tolerant crop, lentil is a major crop in rainfed areas and a suitable candidate for drought stress tolerance research work. Screening for drought tolerance stress under hydroponic conditions at seedling stage with air exposure is an efficient technique to select genotypes with contrasting traits. Transcriptome analysis provides valuable resources, especially for lentil, as here the information on complete genome sequence is not available. Hence, the present studies were carried out.ResultsThis study was undertaken to understand the biochemical mechanisms and transcriptome changes involved in imparting adaptation to drought stress at seedling stage in drought-tolerant (PDL-2) and drought-sensitive (JL-3) cultivars. Among different physiological and biochemical parameters, a significant increase was recorded in proline, glycine betaine contents and activities of SOD, APX and GPX in PDL-2 compared to JL-3while chlorophyll, RWC and catalase activity decreased significantly in JL-3. Transcriptome changes between the PDL-2 and JL-3 under drought stress were evaluated using Illumina HiSeq 2500 platform. Total number of bases ranged from 5.1 to 6.7 Gb. Sequence analysis of control and drought treated cDNA libraries of PDL-2 and JL-3 produced 74032, 75500, 78328 and 81523 contigs, respectively with respective N50 value of 2011, 2008, 2000 and 1991. Differential gene expression of drought treated genotypes along with their controls revealed a total of 11,435 upregulated and 6,934 downregulated transcripts. For functional classification of DEGs, KEGG pathway annotation analysis extracted a total of 413 GO annotation terms where 176 were within molecular process, 128 in cellular and 109 in biological process groups.ConclusionThe transcriptional profiles provide a foundation for deciphering the underlying mechanism for drought tolerance in lentil. Transcriptional regulation, signal transduction and secondary metabolism in two genotypes revealed significant differences at seedling stage under severe drought. Our finding suggests role of candidate genes for improving drought tolerance in lentil.
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Project description:Complexes of Arthrobacter globiformis amine oxidase (AGAO) with the inhibitors benzylhydrazine and tranylcypromine (an antidepressant drug) have been refined at 1.86 and 1.65 A resolution, respectively. Both inhibitors form covalent adducts with the TPQ cofactor. A tyrosine residue, proposed to act as a gate to the AGAO active site, is in its open conformation.
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Project description:Polyamines (PAs) are essential metabolites in plants performing multiple functions during growth and development. Copper-containing amine oxidases (CuAOs) catalyse the catabolism of PAs and in Arabidopsis thaliana are encoded by a gene family. Two mutants of one gene family member, AtCuAO?, showed delayed seed germination, leaf emergence, and flowering time. The height of the primary inflorescence shoot was reduced, and developmental leaf senescence was delayed. Siliques were significantly longer in mutant lines and contained more seeds. The phenotype of AtCuAO? over-expressors was less affected. Before flowering, there was a significant increase in putrescine in AtCuAO? mutant leaves compared to wild type (WT), while after flowering both spermidine and spermine concentrations were significantly higher than in WT leaves. The expression of GA (gibberellic acid) biosynthetic genes was repressed and the content of GA1, GA7, GA8, GA9, and GA20 was reduced in the mutants. The inhibitor of copper-containing amine oxidases, aminoguanidine hydrochloride, mimicked the effect of AtCuAO? mutation on WT seed germination. Delayed germination, reduced shoot height, and delayed flowering in the mutants were rescued by GA3 treatment. These data strongly suggest AtCuAO? is an important gene regulating PA homeostasis, and that a perturbation of PAs affects plant development through a reduction in GA biosynthesis.
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Project description:Copper-containing amine oxidases are found in all the major kingdoms of life. They catalyse the oxidation of organic amines in the presence of molecular dioxygen to aldehydes and hydrogen peroxide. The catalytic centres contain a Cu atom and a topaquinone cofactor formed autocatalytically from a tyrosine residue in the presence of Cu and molecular oxygen. The structure of the Cu-containing amine oxidase from Arthrobacter globiformis, which was previously refined at 1.8 A resolution in space group C2 with unit-cell parameters a = 157.84, b = 63.24, c = 91.98 A, beta = 112.0 degrees [Wilce et al. (1997), Biochemistry, 36, 16116-16133], has been re-refined with newly recorded data at 1.55 A resolution. The structure has also been solved and refined at 2.2 A resolution in a new crystal form, space group C2, with unit-cell parameters a = 158.04, b = 64.06, c = 69.69 A, beta = 111.7 degrees.
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Project description:Amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) is a secreted glycoprotein that catalyzes the degradation of putrescine and histamine. Polyamines and their diamine precursor putrescine are ubiquitous to all organisms and fulfill pivotal functions in cell growth and proliferation. Despite the importance of AOC1 in regulating polyamine breakdown, very little is known about the molecular mechanisms that control its expression. We report here that the Wilms tumor protein, WT1, which is necessary for normal kidney development, activates transcription of the AOC1 gene. Expression of a firefly luciferase reporter under control of the proximal AOC1 promoter was significantly enhanced by co-transfection of a WT1 expression construct. Binding of WT1 protein to a cis-regulatory element in the AOC1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Antisense inhibition of WT1 protein translation strongly reduced Aoc1 transcripts in cultured murine embryonic kidneys and gonads. Aoc1 mRNA levels correlated with WT1 protein in several cell lines. Double immunofluorescent staining revealed a co-expression of WT1 and AOC1 proteins in the developing genitourinary system of mice and rats. Strikingly, induced changes in polyamine homeostasis affected branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-specific manner. These findings suggest that WT1-dependent control of polyamine breakdown, which is mediated by changes in AOC1 expression, has a role in kidney organogenesis.
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Project description:To reveal the chemical changes and geometry changes of active-site residues that cooperate with a reaction is important for understanding the functional mechanism of proteins. Consecutive temporal analyses of enzyme structures have been performed during reactions to clarify structure-based reaction mechanisms. Phenylethylamine oxidase from Arthrobacter globiformis (AGAO) contains a copper ion and topaquinone (TPQ(ox)). The catalytic reaction of AGAO catalyzes oxidative deaminations of phenylethylamine and consists of reductive and oxidative half-reactions. In the reduction step, TPQ(ox) reacts with a phenylethylamine (PEA) substrate giving rise to a topasemiquinone (TPQ(sq)) formed Schiff-base and produces phenylacetaldehyde. To elucidate the mechanism of the reductive half-reaction, an attempt was made to trap the reaction intermediates in order to analyze their structures. The reaction proceeded within the crystals when AGAO crystals were soaked in a PEA solution and freeze-trapped in liquid nitrogen. The reaction stage of each crystal was confirmed by single-crystal microspectrometry, before X-ray diffraction measurements were made of four reaction intermediates. The structure at 15 min after the onset of the reaction was analyzed at atomic resolution, and it was shown that TPQ(ox) and some residues in the substrate channel were alternated via catalytic reductive half-reactions.
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Project description:Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, ?32 Å from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca2+ (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Ca2+ (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Q mutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (?60% WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (?4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+-substrate reduced TPQ (TPQAMQ) Cu+-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu2+-TPQAMQ or destabilizes Cu+-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.
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