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1.
J Biosci ; 452020.
Artículo en Inglés | MEDLINE | ID: mdl-32345784

RESUMEN

The 165,137 bp plasmid pAO1 of Paenarthrobacter nicotinovorans carries the genes of a nicotine catabolic pathway. The genes are organized into several gene modules responsible for the catabolism of L- and D-nicotine to nicotine blue, alpha-ketoglutarate and succinate. Various modules of these genes have been shown to be present in gram-positive (Gram?) soil bacteria. The presence of the identical pAO1 nic-genes on the 288,370 bp plasmid pZXY21 of Arthrobacter sp. ZXY2 (96 percent to 100 percent at the nucleotide level) permitted the identification of the limits of this DNA fragment. At the 5' end of the nic-genes are located the ORFs of two predicted integrases of the tyrosine recombinase family with conserved R, H, R and Y catalytic residues and that of a small transposase with a predicted leucine zipper motive. They are related to Tn554A, Tn554B and Tn554C of Staphylococcus aureus and suggest that the entire nic-genes DNA fragment represents a large catabolic transposon. Surprisingly the nic-genes on pZXY21 were found to be interspersed by mobile elements encoding transposases of various IS families. Insertion of these IS elements disrupts nicotine degradation and divide the nic-genes DNA into potentially new transposons. This finding may illustrate how nicotine catabolic genes can be mobilized and spread by horizontal gene transfer to other soil bacteria.


Asunto(s)
Arthrobacter/enzimología , Arthrobacter/genética , Proteínas Bacterianas/genética , Elementos Transponibles de ADN , Nicotina/metabolismo , Arthrobacter/metabolismo , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos , Transferencia de Gen Horizontal , Genes Bacterianos , Integrasas/genética , Micrococcaceae/genética , Plásmidos , Microbiología del Suelo
2.
Microbiol Res ; 191: 32-7, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27524651

RESUMEN

BLAST analysis of pAO1 ORFs of Arthrobacter nicotinovorans revealed 12 ORFs, including the ORF of a transcriptional regulator, predicted to encode the components of a T4-secretion system involved in bacterial conjugation. These ORFs were conserved and showed synteny among 14 Arthrobacter plasmids. A DNA repeat of about 370 nucleotides was found to be present 5' to the pAO1 ORFs of DUF4192-, DprA- and ParB-like proteins. Similar repeats were present in identical positions on 12 additional Arthrobacter plasmids. The DNA repeats on a particular plasmid are highly identical duplications. The DNA repeats contain alternating GC and AT reach sequences, potential protein DNA-binding sites and purine reach stretches. The sequences end with 5'ATG.AAC3' which results in the amino terminal sequence methionine (M) and asparagine (N) for all predicted DprA, DUF4192 and ParB proteins. The presences of conserved ORFs of a T4-secretion system and of similar DNA repeats suggest that these Arthrobacter plasmids are related and evolved from a common ancestor. The functional significance of the DNA repeats in a coordinated common mechanism of regulation of expression of the dprA-(involved in natural competence), parB- (involved in plasmid partitioning) and duf4192- (unknown function in plasmid life cycle) genes remains to be established.


Asunto(s)
Arthrobacter/genética , Arthrobacter/metabolismo , Conjugación Genética , Secuencia Conservada , ADN Bacteriano/genética , Plásmidos , Secuencias Repetitivas de Ácidos Nucleicos , Composición de Base , Sitios de Unión , Competencia de la Transformación por ADN , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Sistemas de Lectura Abierta , Transporte de Proteínas
3.
Front Chem ; 3: 30, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25954742

RESUMEN

The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD "chaperone." The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

4.
Neurosci Lett ; 591: 41-47, 2015 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-25684246

RESUMEN

6-Hydroxy-l-nicotine (6HLN) is a nicotine metabolite resulted from nicotine degradation within Arthrobacter nicotinovorans with positive effects on spatial memory and oxidative stress damage. In the present study, the effects of 6HLN on spatial memory performance were assessed in scopolamine-treated rats. Scopolamine-induced memory impairments were observed, as measured by the Y-maze and radial arm-maze tasks. Decreased activities of superoxide dismutase, glutathione peroxidase and catalase along with decrease of total content of reduced glutathione were observed in the rat hippocampal homogenates of scopolamine-treated animals as compared with control. Production of malondialdehyde (lipid peroxidation) significantly increased in the rat hippocampal homogenates of scopolamine-treated animals as compared with control, as a consequence of impaired antioxidant enzymes activities. Additionally, in scopolamine-treated rats 6HLN significantly improved memory formation and decreased oxidative stress, suggesting memory-enhancing and antioxidant effects. Therefore, our results suggest that administration of 6HLN ameliorates scopolamine-induced spatial memory impairment by attenuation of the oxidative stress in the rat hippocampus.


Asunto(s)
Enfermedad de Alzheimer/psicología , Hipocampo/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Nicotina/análogos & derivados , Estrés Oxidativo/efectos de los fármacos , Memoria Espacial/efectos de los fármacos , Enfermedad de Alzheimer/metabolismo , Animales , Hipocampo/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Nicotina/farmacología , Ratas Wistar
5.
J Mol Evol ; 77(1-2): 22-30, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23884627

RESUMEN

The 165-kb megaplasmid pAO1 of Arthrobacter nicotinovorans carries two large gene clusters, one involved in nicotine catabolism (nic-gene cluster) and one in carbohydrate utilization (ch-gene cluster). Here, we propose that both gene clusters were acquired by A. nicotinovorans by horizontal gene transfer mediated by pAO1. Protein-protein blast search showed that none of the published Arthrobacter genomes contains nic-genes, but Rhodococcus opacus carries on its chromosome a nic-gene cluster highly similar to that of pAO1. Analysis of the nic-genes in the two species suggested a recombination event between their nic-gene clusters. Apparently, there was a gene exchange between pAO1, or a precursor plasmid, and a nic-gene cluster of an as yet unidentified Arthrobacter specie or other soil bacterium, possibly related to Rhodococcus, leading to the transfer by pAO1 of this catabolic trait to A. nicotinovorans. Analysis of the pAO1 ch-gene cluster revealed a virtually identical counterpart on the chromosome of Arthrobacter phenanthrenivorans. Moreover, the sequence analysis of the genes flanking the ch-gene cluster suggested that it was acquired by pAO1 by Xer-related site directed recombination and transferred via the plasmid to A. nicotinovorans. The G+C content, the level of sequence identity, gene co-linearity of nic- and ch-gene clusters as well as the signs of recombination events clearly supports the notion of pAO1 and its precursor plasmids as vehicles in HGT among Gram + soil bacteria.


Asunto(s)
Arthrobacter/genética , Proteínas Bacterianas/genética , Transferencia de Gen Horizontal , Carácter Cuantitativo Heredable , Arthrobacter/clasificación , Arthrobacter/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos , Orden Génico , Familia de Multigenes , Fenotipo , Filogenia , Plásmidos/genética , Microbiología del Suelo
6.
J. physiol. biochem ; 69(1): 25-34, mar. 2013.
Artículo en Inglés | IBECS | ID: ibc-121984

RESUMEN

Male Wistar rats were subjected to chronic 6-hydroxy-L-nicotine treatment (6HLN, 0.3 mg/kg, i.p., seven consecutive days) and their memory performance was studied by means of Y-maze and radial arm-maze tasks. 6HLN significantly increased spontaneous alternations in Y-maze task and working memory in radial arm-maze task, suggesting effects on short-term memory, without affecting long-term memory, explored by reference memory in radial arm-maze task. In addition, 6HLN increased antioxidant enzymes activity and decreased production of lipid peroxidation, suggesting antioxidant effects. Also, the linear regression between behavioral measures and oxidative stress markers resulted in significant correlations. Therefore, positive effects of 6HLN on spatial memory may occur by antioxidant actions (AU)


Asunto(s)
Animales , Ratas , Nicotina/administración & dosificación , Memoria , Antioxidantes/farmacocinética , Percepción Espacial
7.
Res Microbiol ; 164(1): 22-30, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23063486

RESUMEN

Due to its high abundance, the D-xylose fraction of lignocellulose provides a promising resource for production of various chemicals. Examples of efficient utilization of d-xylose are nevertheless rare, mainly due to the lack of enzymes with suitable properties for biotechnological applications. The genus Arthrobacter, which occupies an ecological niche rich in lignocellulosic materials and containing species with high resistance and tolerance to environmental factors, is a very suitable candidate for finding D-xylose-degrading enzymes with new properties. In this work, the presence of the pAO1 megaplasmid in cells of Arthrobacter nicotinovorans was directly linked to the ability of this microorganism to ferment D-xylose and to sustain longer log growth. Three pAO1 genes (orf32, orf39, orf40) putatively involved in degradation of xylose were identified and cloned, and the corresponding proteins purified and characterized. ORF40 was shown to be a homotetrameric NADP(+)/NAD(+) sugar dehydrogenase with a strong preference for d-xylose; ORF39 is a monomeric aldehyde dehydrogenase with wide substrate specificity and ORF32 is a constitutive expressed transcription factor putatively involved in control of the entire catabolic pathway. Based on analogies with other pentose degradation pathways, a putative xylose oxidative pathway similar to the Weimberg pathway is postulated.


Asunto(s)
Aldehído Oxidasa/genética , Aldehído Oxidasa/metabolismo , Arthrobacter/genética , Arthrobacter/metabolismo , Redes y Vías Metabólicas , Plásmidos/genética , Xilosa/metabolismo , Arthrobacter/crecimiento & desarrollo , Medios de Cultivo , Expresión Génica , Orden Génico , Sistemas de Lectura Abierta , Oxidación-Reducción , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
8.
J Physiol Biochem ; 69(1): 25-34, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22714706

RESUMEN

Male Wistar rats were subjected to chronic 6-hydroxy-L-nicotine treatment (6HLN, 0.3 mg/kg, i.p., seven consecutive days) and their memory performance was studied by means of Y-maze and radial arm-maze tasks. 6HLN significantly increased spontaneous alternations in Y-maze task and working memory in radial arm-maze task, suggesting effects on short-term memory, without affecting long-term memory, explored by reference memory in radial arm-maze task. In addition, 6HLN increased antioxidant enzymes activity and decreased production of lipid peroxidation, suggesting antioxidant effects. Also, the linear regression between behavioral measures and oxidative stress markers resulted in significant correlations. Therefore, positive effects of 6HLN on spatial memory may occur by antioxidant actions.


Asunto(s)
Arthrobacter/química , Encéfalo/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Memoria a Corto Plazo/efectos de los fármacos , Nicotina/análogos & derivados , Estrés Oxidativo/efectos de los fármacos , Animales , Encéfalo/metabolismo , Glutatión Peroxidasa/metabolismo , Inyecciones Intraperitoneales , Peroxidación de Lípido/efectos de los fármacos , Masculino , Malondialdehído/metabolismo , Aprendizaje por Laberinto/fisiología , Memoria a Largo Plazo/efectos de los fármacos , Memoria a Largo Plazo/fisiología , Memoria a Corto Plazo/fisiología , Nicotina/aislamiento & purificación , Nicotina/farmacología , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo
9.
Res Microbiol ; 162(3): 285-91, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21288482

RESUMEN

Gram-positive soil bacteria Arthrobacter nicotinovorans, Nocardioides sp. JS614 and Rhodococcus opacus were shown to contain similarly organized clusters of homologous genes for nicotine catabolism. An uncharacterized gene of a predicted nitrilase within these gene clusters was cloned from A. nicotinovorans and biochemical data unexpectedly showed that the protein exhibited ω-amidase activity toward α-ketoglutaramate. Structural modelling of the protein suggested the presence of the catalytic triad Cys-Glu-Lys, characteristic of this class of enzymes, and supported α-ketoglutaramate as substrate. A-ketoglutaramate could be generated by hydrolytic cleavage of the C-N bond of the trihydroxypyridine ring produced by nicotine catabolism in these bacteria. This ω-amidase, together with glutamate dehydrogenase, may form a physiologically relevant enzyme couple, leading to transformation of metabolically inert α-ketoglutaramate derived from trihydroxypyridine into glutamate, a central compound of nitrogen metabolism.


Asunto(s)
Actinomycetales/genética , Amidohidrolasas/genética , Arthrobacter/genética , Ácidos Cetoglutáricos/metabolismo , Redes y Vías Metabólicas/genética , Nicotina/metabolismo , Rhodococcus/genética , Actinomycetales/metabolismo , Amidohidrolasas/metabolismo , Arthrobacter/metabolismo , Dominio Catalítico , Orden Génico , Modelos Moleculares , Familia de Multigenes , Estructura Terciaria de Proteína , Rhodococcus/metabolismo
10.
Microbiology (Reading) ; 155(Pt 6): 1866-1877, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19443550

RESUMEN

The mechanism by which l-nicotine is taken up by bacteria that are able to grow on it is unknown. Nicotine degradation by Arthrobacter nicotinovorans, a Gram-positive soil bacterium, is linked to the presence of the catabolic megaplasmid pAO1. l-[(14)C]Nicotine uptake assays with A. nicotinovorans showed transport of nicotine across the cell membrane to be energy-independent and saturable with a K(m) of 6.2+/-0.1 microM and a V(max) of 0.70+/-0.08 micromol min(-1) (mg protein)(-1). This is in accord with a mechanism of facilitated diffusion, driven by the nicotine concentration gradient. Nicotine uptake was coupled to its intracellular degradation, and an A. nicotinovorans strain unable to degrade nicotine (pAO1(-)) showed no nicotine import. However, when the nicotine dehydrogenase genes were expressed in this strain, import of l-[(14)C]nicotine took place. A. nicotinovorans pAO1(-) and Escherichia coli were also unable to import 6-hydroxy-l-nicotine, but expression of the 6-hydroxy-l-nicotine oxidase gene allowed both bacteria to take up this compound. l-Nicotine uptake was inhibited by d-nicotine, 6-hydroxy-l-nicotine and 2-amino-l-nicotine, which may indicate transport of these nicotine derivatives by a common permease. Attempts to correlate nicotine uptake with pAO1 genes possessing similarity to amino acid transporters failed. In contrast to the situation at the blood-brain barrier, nicotine transport across the cell membrane by these bacteria was not by passive diffusion or active transport but by facilitated diffusion.


Asunto(s)
Arthrobacter/metabolismo , Escherichia coli/metabolismo , Difusión Facilitada , Nicotina/análogos & derivados , Arthrobacter/ultraestructura , Transporte Biológico Activo , Membrana Celular/metabolismo , Escherichia coli/ultraestructura , Proteínas de Transporte de Membrana/metabolismo , Nicotina/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Plásmidos/genética , Especificidad por Sustrato
11.
Int J Biol Macromol ; 42(5): 455-62, 2008 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-18423846

RESUMEN

The precursor of the rat mitochondrial flavoenzyme dimethylglycine dehydrogenase (Me(2)GlyDH) has been produced in Escherichia coli as a C-terminally 6-His-tagged fusion protein, purified by one-step affinity chromatography and identified by ESI-MS/MS. It was correctly processed into its mature form upon incubation with solubilized rat liver mitoplasts. The purified precursor was mainly in its apo-form as demonstrated by immunological and fluorimetric detection of covalently bound flavin adenine dinucleotide (FAD). Results described here definitively demonstrate that: (i) covalent attachment of FAD to Me(2)GlyDH apoenzyme can proceed in vitro autocatalytically, without third reactants; (ii) the removal of mitochondrial presequence by mitochondrial processing peptidase is not required for covalent autoflavinylation.


Asunto(s)
Dimetilglicina-Deshidrogenasa/aislamiento & purificación , Dimetilglicina-Deshidrogenasa/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/aislamiento & purificación , Proteínas Mitocondriales/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Clonación Molecular , Dimetilglicina-Deshidrogenasa/química , Dimetilglicina-Deshidrogenasa/genética , Expresión Génica , Espectrometría de Masas , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrofotometría
12.
Arch Microbiol ; 189(5): 511-7, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18071673

RESUMEN

A virtually identical nicotine catabolic pathway including the heterotrimeric molybdenum enzyme nicotine and 6-hydroxy-pseudo-oxynicotine dehydrogenase, 6-hydroxy-L: -nicotine oxidase, 2,6-dihydroxy-pseudo-oxynicotine hydrolase, and 2,6-dihydroxypyridine hydroxylase have been identified in A. nicotinovorans and Nocardioides sp. JS614. Enzymes catalyzing the same reactions and similar protein antigens were detected in the extracts of the two microorganisms. Nicotine blue and methylamine, two end products of nicotine catabolism were detected in the growth medium of both bacterial species. Nicotine catabolic genes are clustered on pAO1 in A. nicotinovorans, but located chromosomally in Nocardioides sp. JS614.


Asunto(s)
Actinomycetales/enzimología , Arthrobacter/enzimología , Oxigenasas de Función Mixta/metabolismo , Nicotina/metabolismo , Actinomycetales/genética , Secuencia de Aminoácidos , Arthrobacter/genética , Western Blotting , Mapeo Cromosómico , Cromosomas Bacterianos , Hidroxilación , Metilaminas/metabolismo , Familia de Multigenes , Sistemas de Lectura Abierta , Plásmidos , Piridonas/metabolismo
13.
Microbiology (Reading) ; 153(Pt 5): 1546-1555, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17464069

RESUMEN

The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.


Asunto(s)
Arthrobacter/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Nicotina/metabolismo , 2,4-Dinitrofenol/farmacología , Secuencia de Aminoácidos , Aminobutiratos/metabolismo , Arthrobacter/efectos de los fármacos , Proteínas Bacterianas/genética , Radioisótopos de Carbono/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Eliminación de Gen , Prueba de Complementación Genética , Ionóforos/farmacología , Marcaje Isotópico , Metilaminas/metabolismo , Datos de Secuencia Molecular , Nigericina/farmacología , Nitrilos/farmacología , Alineación de Secuencia , Desacopladores/farmacología
14.
Appl Environ Microbiol ; 73(8): 2479-85, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17293530

RESUMEN

An NAD(P)H-nicotine blue (quinone) oxidoreductase was discovered as a member of the nicotine catabolic pathway of Arthrobacter nicotinovorans. Transcriptional analysis and electromobility shift assays showed that the enzyme gene was expressed in a nicotine-dependent manner under the control of the transcriptional activator PmfR and thus was part of the nicotine regulon of A. nicotinovorans. The flavin mononucleotide-containing enzyme uses NADH and, with lower efficiency, NADPH to reduce, by a two-electron transfer, nicotine blue to the nicotine blue leuco form (hydroquinone). Besides nicotine blue, several other quinones were reduced by the enzyme. The NAD(P)H-nicotine blue oxidoreductase may prevent intracellular one-electron reductions of nicotine blue which may lead to semiquinone radicals and potentially toxic reactive oxygen species.


Asunto(s)
Arthrobacter/fisiología , Nicotina/metabolismo , Estrés Oxidativo , Quinona Reductasas/genética , Quinona Reductasas/metabolismo , Regulón , Arthrobacter/enzimología , Arthrobacter/genética , Clonación Molecular , Coenzimas/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Mononucleótido de Flavina/farmacología , Regulación Bacteriana de la Expresión Génica/fisiología , Hidroquinonas/metabolismo , Modelos Biológicos , NAD/metabolismo , NADP/metabolismo , Oxidación-Reducción , Quinona Reductasas/aislamiento & purificación , Quinona Reductasas/fisiología , ARN Bacteriano/biosíntesis , ARN Bacteriano/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Especificidad por Sustrato
15.
J Mol Biol ; 367(2): 409-18, 2007 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-17275835

RESUMEN

The enzyme 2,6-dihydroxy-pseudo-oxynicotine hydrolase from the nicotine-degradation pathway of Arthrobacter nicotinovorans was crystallized and the structure was determined by an X-ray diffraction analysis at 2.1 A resolution. The enzyme belongs to the alpha/beta-hydrolase family as derived from the chain-fold and from the presence of a catalytic triad with its oxyanion hole at the common position. This relationship assigns a pocket lined by the catalytic triad as the active center. The asymmetric unit contains two C(2)-symmetric dimer molecules, each adopting a specific conformation. One dimer forms a more spacious active center pocket and the other a smaller one, suggesting an induced-fit. All of the currently established C-C bond cleaving alpha/beta-hydrolases are from bacterial meta-cleavage pathways for the degradation of aromatic compounds and cover their active center with a 40 residue lid placed between two adjacent strands of the beta-sheet. In contrast, the reported enzyme shields its active center with a 110 residue N-terminal domain, which is absent in the meta-cleavage hydrolases. Since neither the substrate nor an analogue could be bound in the crystals, the substrate was modeled into the active center using the oxyanion hole as a geometric constraint. The model was supported by enzymatic activity data of 11 point mutants and by the two dimer conformations suggesting an induced-fit. Moreover, the model assigned a major role for the large N-terminal domain that is specific to the reported enzyme. The proposal is consistent with the known data for the meta-cleavage hydrolases although it differs in that the reaction does not release alkenes but a hetero-aromatic compound in a retro-Friedel-Crafts acylation. Because the hydrolytic water molecule can be assigned to a geometrically suitable site that can be occupied in the presence of the substrate, the catalytic triad may not form a covalent acyl-enzyme intermediate but merely support a direct hydrolysis.


Asunto(s)
Arthrobacter/enzimología , Proteínas Bacterianas/química , Hidrolasas/química , Modelos Moleculares , Nicotina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dimerización , Hidrolasas/genética , Hidrolasas/metabolismo , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de Proteína , Agua/química
16.
Appl Environ Microbiol ; 72(7): 5126-31, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16820521

RESUMEN

Two Arthrobacter nicotinovorans molybdenum enzymes hydroxylate the pyridine ring of nicotine. Molybdopterin cytosine dinucleotide (MCD) was determined to be a cofactor of these enzymes. A mobA gene responsible for the formation of MCD could be identified and its function shown to be required for assembly of the heterotrimeric molybdenum enzymes.


Asunto(s)
Arthrobacter/enzimología , Proteínas Bacterianas/genética , Nucleótidos de Citosina/biosíntesis , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Holoenzimas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Transactivadores/genética , Arthrobacter/genética , Nucleótidos de Citosina/genética , Dimerización , Pterinas
17.
FEBS J ; 273(7): 1528-36, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16689938

RESUMEN

New enzymes of nicotine catabolism instrumental in the detoxification of the tobacco alkaloid by Arthrobacter nicotinovorans pAO1 have been identified and characterized. Nicotine breakdown leads to the formation of nicotine blue from the hydroxylated pyridine ring and of gamma-N-methylaminobutyrate (CH(3)-4-aminobutyrate) from the pyrrolidine ring of the molecule. Surprisingly, two alternative pathways for the final steps in the catabolism of CH(3)-4-aminobutyrate could be identified. CH(3)-4-aminobutyrate may be demethylated to gamma-N-aminobutyrate by the recently identified gamma-N-methylaminobutyrate oxidase. In an alternative pathway, an amine oxidase with noncovalently bound FAD and of novel substrate specificity removed methylamine from CH(3)-4-aminobutyrate with the formation of succinic semialdehyde. Succinic semialdehyde was converted to succinate by a NADP(+)-dependent succinic semialdehyde dehydrogenase. Succinate may enter the citric acid cycle completing the catabolism of the pyrrolidine moiety of nicotine. Expression of the genes of these enzymes was dependent on the presence of nicotine in the growth medium. Thus, two enzymes of the nicotine regulon, gamma-N-methylaminobutyrate oxidase and amine oxidase share the same substrate. The K(m) of 2.5 mM and k(cat) of 1230 s(-1) for amine oxidase vs. K(m) of 140 microM and k(cat) of 800 s(-1) for gamma-N-methylaminobutyrate oxidase, determined in vitro with the purified recombinant enzymes, may suggest that demethylation predominates over deamination of CH(3)-4-aminobutyrate. However, bacteria grown on [(14)C]nicotine secreted [(14)C]methylamine into the medium, indicating that the pathway to succinate is active in vivo.


Asunto(s)
Arthrobacter/metabolismo , Proteínas Bacterianas/metabolismo , Nicotina/metabolismo , Oxidorreductasas/metabolismo , Aminobutiratos/metabolismo , Arthrobacter/genética , Proteínas Bacterianas/genética , Humanos , Metilaminas/metabolismo , Estructura Molecular , Nicotina/química , Oxidorreductasas/genética , Plásmidos/genética , Plásmidos/metabolismo , Succionato-Semialdehído Deshidrogenasa/genética , Succionato-Semialdehído Deshidrogenasa/metabolismo
18.
Appl Microbiol Biotechnol ; 69(5): 493-8, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16333621

RESUMEN

Several bacterial species are adapted to nicotine, the main alkaloid produced by the tobacco plant, as growth substrate. A general outline of nicotine catabolism by these bacteria is presented, followed by an emphasis on new insights based on molecular biology and biochemical work obtained with the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. Its 165-kb sequence revealed the genetic structure of nicotine catabolism and allowed the assignment of new enzyme activities to specific gene products, which extends the known biochemical steps of this pathway. Potential implications of the progress in our understanding of bacterial breakdown of nicotine for biotechnological applications are discussed.


Asunto(s)
Arthrobacter/metabolismo , Nicotina/metabolismo , Plásmidos , Arthrobacter/genética , Biodegradación Ambiental
19.
J Bacteriol ; 187(24): 8516-9, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16321959

RESUMEN

The enzyme catalyzing the hydrolytic cleavage of 2,6-dihydroxypseudooxynicotine to 2,6-dihydroxypyridine and gamma-N-methylaminobutyrate was found to be encoded on pAO1 of Arthrobacter nicotinovorans. The new enzyme answers an old question about nicotine catabolism and may be the first C--C bond hydrolase that is involved in the biodegradation of a heterocyclic compound.


Asunto(s)
Arthrobacter/enzimología , Hidrolasas/química , Hidrolasas/metabolismo , Nicotina/metabolismo , Alcaloides/metabolismo , Arthrobacter/metabolismo , Biodegradación Ambiental , Cromatografía Líquida de Alta Presión , Clonación Molecular , Orden Génico , Compuestos Heterocíclicos/metabolismo , Nicotina/análogos & derivados , Sistemas de Lectura Abierta , Plásmidos/genética , Piridinas/metabolismo
20.
Appl Environ Microbiol ; 71(12): 8920-4, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16332890

RESUMEN

The first inducible Arthrobacter overexpression system, based on the promoter/operator and the repressor of the 6-D-hydroxynicotine oxidase gene of Arthrobacter nicotinovorans, is described here. Nicotine-dependent overproduction and affinity purification of recombinant proteins are presented. The system will allow the production of complex enzymes and genetic complementation studies in Arthrobacter species.


Asunto(s)
Arthrobacter/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Nicotina/farmacología , Plásmidos/genética , Arthrobacter/clasificación , Secuencia de Bases , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Datos de Secuencia Molecular , Plásmidos/efectos de los fármacos , Mapeo Restrictivo
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