| 1. |
Sandu C,
Chiribau CB,
Brandsch R,
( 2003 ) Characterization of HdnoR, the transcriptional repressor of the 6-hydroxy-D-nicotine oxidase gene of Arthrobacter nicotinovorans pAO1, and its DNA-binding activity in response to L- and D-nicotine Derivatives. PMID : 14534317 : DOI : 10.1074/jbc.M307797200 Abstract >>
Utilization of L-nicotine as growth substrate by Arthrobacter nicotinovorans pAO1 starts with hydroxylation of the pyridine ring at C6. Next, the pyrrolidine ring is oxidized by 6-hydroxy-L-nicotine oxidase, which acts strictly stereo-specific on the L-enantiomer. Surprisingly, L-nicotine also induces the synthesis of a 6-hydroxy-d-nicotine-specific oxidase in the bacteria. Genes of nicotine-degrading enzymes are located on the catabolic plasmid pAO1. The pAO1 sequence revealed that the 6-hydroxy-D-nicotine oxidase gene is flanked by two open reading frames with a similarity to amino acid permeases and a divergently transcribed open reading frame with a similarity to proteins of the tetracycline repressor TetR family. Reverse transcription PCR and primer extension analysis of RNA transcripts isolated from A. nicotinovorans pAO1 indicated that the 6-hydroxy-D-nicotine oxidase gene represents a transcriptional unit. DNA electromobility shift assays established that the purified TetR-similar protein represents the 6-hydroxy-D-nicotine oxidase gene repressor HdnoR and binds to the 6-hydroxy-D-nicotine oxidase gene operator with a Kd of 21 nM. The enantiomers 6-hydroxy-D- and 6-hydroxy-L-nicotine acted in vitro as inducers. In vivo analysis of 6-hydroxy-D-nicotine oxidase gene transcripts from bacteria grown with L- and D-nicotine confirmed this conclusion. The poor discrimination by HdnoR between the 6-hydroxy-L- and 6-hydroxy-D-nicotine enantiomers explains the presence of the 6-hydroxy-D-nicotine-specific enzyme in bacteria grown on L-nicotine.
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2. |
Igloi GL,
Brandsch R,
( 2003 ) Sequence of the 165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system. PMID : 12618462 : DOI : 10.1128/jb.185.6.1976-1986.2003 PMC : PMC150138 Abstract >>
The 165-kb catabolic plasmid pAO1 enables the gram-positive soil bacterium Arthrobacter nicotinovorans to grow on the tobacco alkaloid L-nicotine. The 165,137-nucleotide sequence, with an overall G+C content of 59.7%, revealed, besides genes and open reading frames (ORFs) for nicotine degradation, a complete set of ORFs for enzymes essential for the biosynthesis of the molybdenum dinucleotide cofactor, as well as ORFs related to uptake and utilization of carbohydrates, sarcosine, and amino acids. Of the 165 ORFs, approximately 50% were related to metabolic functions. pAO1 conferred to A. nicotinovorans the ability to take up L-[(14)C]nicotine from the medium, with an K(m) of 5.6 +/- 2.2 micro M. ORFs of putative nicotine transporters formed a cluster with the gene of the D-nicotine-specific 6-hydroxy-D-nicotine oxidase. ORFs related to replication, chromosome partitioning, and natural transformation functions (dprA) were identified on pAO1. Few ORFs showed similarity to known conjugation-promoting proteins, but pAO1 could be transferred by conjugation to a pAO1-negative strain at a rate of 10(-2) to 10(-3) per donor. ORFs with no known function represented approximately 35% of the pAO1 sequence. The positions of insertion sequence elements and composite transposons, corroborated by the G+C content of the pAO1 sequence, suggest a modular composition of the plasmid.
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3. |
Baitsch D,
Sandu C,
Brandsch R,
Igloi GL,
( 2001 ) Gene cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: cloning, purification, and characterization of 2,6-dihydroxypyridine 3-hydroxylase. PMID : 11514508 : DOI : 10.1128/jb.183.18.5262-5267.2001 PMC : PMC95407 Abstract >>
A 27,690-bp gene cluster involved in the degradation of the plant alkaloid nicotine was characterized from the plasmid pAO1 of Arthrobacter nicotinovorans. The genes of the heterotrimeric, molybdopterin cofactor (MoCo)-, flavin adenine dinucleotide (FAD)-, and [Fe-S] cluster-dependent 6-hydroxypseudooxynicotine (ketone) dehydrogenase (KDH) were identified within this cluster. The gene of the large MoCo subunit of KDH was located 4,266 bp from the FAD and [Fe-S] cluster subunit genes. Deduced functions of proteins encoded by open reading frames (ORFs) of the cluster were correlated to individual steps in nicotine degradation. The gene for 2,6-dihydroxypyridine 3-hydroxylase was cloned and expressed in Escherichia coli. The purified homodimeric enzyme of 90 kDa contained 2 mol of tightly bound FAD per mol of dimer. Enzyme activity was strictly NADH-dependent and specific for 2,6-dihydroxypyridine. 2,3-Dihydroxypyridine and 2,6-dimethoxypyridine acted as irreversible inhibitors. Additional ORFs were shown to encode hypothetical proteins presumably required for holoenzyme assembly, interaction with the cell membrane, and transcriptional regulation, including a MobA homologue predicted to be specific for the synthesis of the molybdopterin cytidine dinucleotide cofactor.
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4. |
Ganas P,
Mihasan M,
Igloi GL,
Brandsch R,
( 2007 ) A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans. PMID : 17464069 : DOI : 10.1099/mic.0.2006/004234-0 Abstract >>
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.
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5. |
Schleberger C,
Sachelaru P,
Brandsch R,
Schulz GE,
( 2007 ) Structure and action of a C-C bond cleaving alpha/beta-hydrolase involved in nicotine degradation. PMID : 17275835 : DOI : 10.1016/j.jmb.2006.12.068 Abstract >>
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.
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6. |
Mihasan M,
Chiribau CB,
Friedrich T,
Artenie V,
Brandsch R,
( 2007 ) An NAD(P)H-nicotine blue oxidoreductase is part of the nicotine regulon and may protect Arthrobacter nicotinovorans from oxidative stress during nicotine catabolism. PMID : 17293530 : DOI : 10.1128/AEM.02668-06 PMC : PMC1855579 Abstract >>
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.
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7. |
Sachelaru P,
Schiltz E,
Brandsch R,
( 2006 ) A functional mobA gene for molybdopterin cytosine dinucleotide cofactor biosynthesis is required for activity and holoenzyme assembly of the heterotrimeric nicotine dehydrogenases of Arthrobacter nicotinovorans. PMID : 16820521 : DOI : 10.1128/AEM.00437-06 PMC : PMC1489357 Abstract >>
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.
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8. |
Chiribau CB,
Mihasan M,
Ganas P,
Igloi GL,
Artenie V,
Brandsch R,
( 2006 ) Final steps in the catabolism of nicotine. PMID : 16689938 : DOI : 10.1111/j.1742-4658.2006.05173.x Abstract >>
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.
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9. |
Brandsch R,
( 2006 ) Microbiology and biochemistry of nicotine degradation. PMID : 16333621 : DOI : 10.1007/s00253-005-0226-0 Abstract >>
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.
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10. |
Sachelaru P,
Schiltz E,
Igloi GL,
Brandsch R,
( 2005 ) An alpha/beta-fold C--C bond hydrolase is involved in a central step of nicotine catabolism by Arthrobacter nicotinovorans. PMID : 16321959 : DOI : 10.1128/JB.187.24.8516-8519.2005 PMC : PMC1317030 Abstract >>
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.
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11. |
Gao B,
Gupta RS,
( 2005 ) Conserved indels in protein sequences that are characteristic of the phylum Actinobacteria. PMID : 16280504 : DOI : 10.1099/ijs.0.63785-0 Abstract >>
Gram-positive bacteria with a high G+C content are currently recognized as a distinct phylum, Actinobacteria, on the basis of their branching in 16S rRNA trees. Except for an insert in the 23S rRNA, there are no unique biochemical or molecular characteristics known at present that can distinguish this group from all other bacteria. In this work, three conserved indels (i.e. inserts or deletions) are described in three widely distributed proteins that are distinctive characteristics of the Actinobacteria and are not found in any other groups of bacteria. The identified signatures are a 2 aa deletion in cytochrome-c oxidase subunit 1 (Cox1), a 4 aa insert in CTP synthetase and a 5 aa insert in glutamyl-tRNA synthetase (GluRS). Additionally, the actinobacterial specificity of the large insert in the 23S rRNA was also tested. Using primers designed for conserved regions flanking these signatures, fragments of most of these genes were amplified from 23 actinobacterial species, covering many different families and orders, for which no sequence information was previously available. All the 61 sequenced fragments, except two in GluRS, were found to contain the indicated signatures. The presence of these signatures in various species from 20 families within this phylum provides evidence that they are likely distinctive characteristics of the entire phylum, which were introduced in a common ancestor of this group. The absence of all four of these signatures in Symbiobacterium thermophilum suggests that this species, which is distantly related to other actinobacteria in 16S rRNA and CTP synthetase trees, may not be a part of the phylum Actinobacteria. The identified signatures provide novel molecular means for defining and circumscribing the phylum Actinobacteria. Functional studies on them should prove helpful in understanding novel biochemical and physiological characteristics of this group of bacteria.
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12. |
Aislabie J,
Bej AK,
Ryburn J,
Lloyd N,
Wilkins A,
( 2005 ) Characterization of Arthrobacter nicotinovorans HIM, an atrazine-degrading bacterium, from agricultural soil New Zealand. PMID : 16329913 : DOI : 10.1016/j.femsec.2004.11.012 Abstract >>
Arthrobacter nicotinovorans HIM was isolated directly from an agricultural sandy dune soil 6 months after a single application of atrazine. It grew in minimal medium with atrazine as sole nitrogen source but was unable to mineralize 14C-ring-labelled atrazine. Atrazine was degraded to cyanuric acid. In addition to atrazine the bacterium degraded simazine, terbuthylazine, propazine, cyanazine and prometryn but was unable to grow on terbumeton. When added to soil, A. nicotinovorans HIM did enhance mineralization of 14C-ring-labelled atrazine and simazine, in combination with naturally occurring cyanuric acid degrading microbes resident in the soil. Using PCR, the atrazine-degradation genes atzABC were identified in A. nicotinovorans HIM. Cloning of the atzABC genes revealed significant homology (>99%) with the atrazine degradation genes of Pseudomonas sp. strain ADP. The atrazine degradation genes were held on a 96 kbp plasmid.
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13. |
Chiribau CB,
Sandu C,
Igloi GL,
Brandsch R,
( 2005 ) Characterization of PmfR, the transcriptional activator of the pAO1-borne purU-mabO-folD operon of Arthrobacter nicotinovorans. PMID : 15838033 : DOI : 10.1128/JB.187.9.3062-3070.2005 PMC : PMC1082840 Abstract >>
Nicotine catabolism by Arthrobacter nicotinovorans is linked to the presence of the megaplasmid pAO1. Genes involved in this catabolic pathway are arranged on the plasmid into gene modules according to function. During nicotine degradation gamma-N-methylaminobutyrate is formed from the pyrrolidine ring of nicotine. Analysis of the pAO1 open reading frames (ORF) resulted in identification of the gene encoding a demethylating gamma-N-methylaminobutyrate oxidase (mabO). This gene was shown to form an operon with purU- and folD-like genes. Only in bacteria grown in the presence of nicotine could transcripts of the purU-mabO-folD operon be detected, demonstrating that this operon constitutes part of the pAO1 nicotine regulon. Its transcriptional start site was determined by primer extension analysis. Transcription of the operon was shown to be controlled by a new transcriptional regulator, PmfR, the product of a gene that is transcribed divergently from the purU, mabO, and folD genes. PmfR was purified, and electromobility shift assays and DNase I-nuclease digestion experiments were used to determine that its DNA binding site is located between -48 and -88 nucleotides upstream of the transcriptional start site of the operon. Disruption of pmfR by homologous recombination with a chloramphenicol resistance cassette demonstrated that PmfR acts in vivo as a transcriptional activator. Mutagenesis of the PmfR target DNA suggested that the sequence GTTT-14 bp-AAAC is the core binding site of the regulator upstream of the -35 promoter region of the purU-mabO-folD operon.
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14. |
Koetter JW,
Schulz GE,
( 2005 ) Crystal structure of 6-hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans. PMID : 16095622 : DOI : 10.1016/j.jmb.2005.07.041 Abstract >>
The crystal structure of 6-hydroxy-d-nicotine oxidase (EC 1.5.3.6) was solved by X-ray diffraction analysis in three crystal forms at resolutions up to 1.9 A. The enzyme is monomeric in solution and also in the mother liquor but formed disulfide-dimers in all crystals. It belongs to the p-cresol methylhydroxylase-vanillyl-alcohol oxidase family and contains an FAD covalently bound to the polypeptide. The covalent bond of this enzyme was the first for which a purely autocatalytic formation had been shown. In contrast to previous reports, the bond does not involve N(epsilon2) (N3) of His72 but the N(delta1) (N1) atom. The geometry of this reaction is proposed and the autoflavinylation is discussed in the light of homologous structures. The enzyme is specific for 6-hydroxy-D-nicotine and is inhibited by the L-enantiomer. This observation was verified by modeling enzyme-substrate and enzyme-inhibitor complexes, which also showed the geometry of the catalyzed reaction. The binding models indicated that the deprotonation of the substrate rather than the hydride transfer is the specificity-determining step. The functionally closely related 6-hydroxy-L-nicotine oxidase processing the L-enantiomer is sequence-related to the greater glutathione reductase family with quite a different chainfold. A model of this "sister enzyme" derived from known homologous structures suggests that the reported L-substrate specificity and D-enantiomer inhibition are also determined by the location of the deprotonating base.
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15. |
Chiribau CB,
Sandu C,
Fraaije M,
Schiltz E,
Brandsch R,
( 2004 ) A novel gamma-N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1. PMID : 15606755 : DOI : 10.1111/j.1432-1033.2004.04432.x Abstract >>
Nicotine catabolism, linked in Arthrobacter nicotinovorans to the presence of the megaplasmid pAO1, leads to the formation of gamma-N-methylaminobutyrate from the pyrrolidine ring of the alkaloid. Until now the metabolic fate of gamma-N-methylaminobutyrate has been unknown. pAO1 carries a cluster of ORFs with similarity to sarcosine and dimethylglycine dehydrogenases and oxidases, to the bifunctional enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase and to formyltetrahydrofolate deformylase. We cloned and expressed the gene carrying the sarcosine dehydrogenase-like ORF and showed, by enzyme activity, spectrophotometric methods and identification of the reaction product as gamma-aminobutyrate, that the predicted 89 395 Da flavoprotein is a demethylating gamma-N-methylaminobutyrate oxidase. Site-directed mutagenesis identified His67 as the site of covalent attachment of FAD and confirmed Trp66 as essential for FAD binding, for enzyme activity and for the spectral properties of the wild-type enzyme. A Km of 140 microm and a kcat of 800 s(-1) was determined when gamma-N-methylaminobutyrate was used as the substrate. Sarcosine was also turned over by the enzyme, but at a rate 200-fold slower than gamma-N-methylaminobutyrate. This novel enzyme activity revealed that the first step in channelling the gamma-N-methylaminobutyrate generated from nicotine into the cell metabolism proceeds by its oxidative demethylation.
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16. |
Kachalova G,
Decker K,
Holt A,
Bartunik HD,
( 2011 ) Crystallographic snapshots of the complete reaction cycle of nicotine degradation by an amine oxidase of the monoamine oxidase (MAO) family. PMID : 21383134 : DOI : 10.1073/pnas.1016684108 PMC : PMC3064382 Abstract >>
FAD-linked oxidases constitute a class of enzymes which catalyze dehydrogenation as a fundamental biochemical reaction, followed by reoxidation of reduced flavin. Here, we present high-resolution crystal structures showing the flavoenzyme 6-hydroxy-l-nicotine oxidase in action. This enzyme was trapped during catalytic degradation of the native substrate in a sequence of discrete reaction states corresponding to the substrate-reduced enzyme, a complex of the enzyme with the intermediate enamine product and formation of the final aminoketone product. The inactive d-stereoisomer binds in mirror symmetry with respect to the catalytic axis, revealing absolute stereospecificity of hydrogen transfer to the flavin. The structural data suggest deprotonation of the substrate when bound at the active site, an overall binary complex mechanism and oxidation by direct hydride transfer. The amine nitrogen has a critical role in the dehydrogenation step and may activate carbocation formation at the �\-carbon via delocalization from the lone pair to �m* C(�\)-H. Enzymatically assisted hydrolysis of the intermediate product occurs at a remote (P site) cavity. Substrate entry and product exit follow different paths. Structural and kinetic data suggest that substrate can also bind to the reduced enzyme, associated with slower reoxidation as compared to the rate of reoxidation of free enzyme. The results are of general relevance for the mechanisms of flavin amine oxidases.
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17. |
Cobzaru C,
Ganas P,
Mihasan M,
Schleberger P,
Brandsch R,
( 2011 ) Homologous gene clusters of nicotine catabolism, including a new �s-amidase for �\-ketoglutaramate, in species of three genera of Gram-positive bacteria. PMID : 21288482 : DOI : 10.1016/j.resmic.2011.01.001 Abstract >>
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 �s-amidase activity toward �\-ketoglutaramate. Structural modelling of the protein suggested the presence of the catalytic triad Cys-Glu-Lys, characteristic of this class of enzymes, and supported �\-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 �s-amidase, together with glutamate dehydrogenase, may form a physiologically relevant enzyme couple, leading to transformation of metabolically inert �\-ketoglutaramate derived from trihydroxypyridine into glutamate, a central compound of nitrogen metabolism.
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18. |
Kachalova GS,
Bourenkov GP,
Mengesdorf T,
Schenk S,
Maun HR,
Burghammer M,
Riekel C,
Decker K,
Bartunik HD,
( 2010 ) Crystal structure analysis of free and substrate-bound 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans. PMID : 20006620 : DOI : 10.1016/j.jmb.2009.12.009 Abstract >>
The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-L-nicotine oxidase (6HLNO) and 6-hydroxy-D-nicotine oxidase, which act with absolute stereospecificity on the L- and D-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 A resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit-subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-L-nicotine at 2.05 A resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-D-nicotine oxidase, based on models of complexes with the D-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.
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19. |
Treiber N,
Schulz GE,
( 2008 ) Structure of 2,6-dihydroxypyridine 3-hydroxylase from a nicotine-degrading pathway. PMID : 18440023 : DOI : 10.1016/j.jmb.2008.03.032 Abstract >>
The enzyme 2,6-dihydroxypyridine-3-hydroxylase catalyzes the sixth step of the nicotine degradation pathway in Arthrobacter nicotinovorans. The enzyme was produced in Escherichia coli, purified and crystallized. The crystal structure was solved at 2.6 A resolution, revealing a significant structural relationship with the family of FAD-dependent aromatic hydroxylases, but essentially no sequence homology. The structure was aligned with those of the established family members, showing that the FAD molecules are bound at virtually identical locations. The reported enzyme is a dimer like most other family members, but its dimerization contact differs from the others. The binding position of NAD(P)H to this enzyme family is not clear. Since the reported enzyme accepts only NADH for flavin reduction in contrast to the other established members using NADPH, we searched through the structural alignment and found an indication for the position of the 2'-phosphate of NADPH that is in general agreement with mutational studies on a related enzyme, but contradicts a crystal soaking experiment. Using a bound glycerol molecule and the known substrate positions of three related enzymes as a guide, the substrate 2,6-dihydroxypyridine was placed into the active center. The access to the binding site is discussed. The new active center geometry introduces constraints that render some reaction scenarios more likely than others. It suggests that flavin is reduced at its out-position and then drawn into its in-position, where it binds molecular oxygen. The geometry is consistent with the proposal that peroxy-flavin is protonated by the solvent to yield the electrophilic hydroperoxy-flavin. The substrate is activated by two buried histidines but there is no appropriate base to store the surplus proton of the hydroxylated carbon atom. The implications of this problem are discussed.
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20. |
Sone T,
Haraguchi Y,
Kuwahara A,
Ose T,
Takano M,
Abe A,
Tanaka M,
Tanaka I,
Asano K,
( 2015 ) Structural characterization reveals the keratinolytic activity of an arthrobacter nicotinovorans protease. PMID : 25256266 : Abstract >>
Elevated cadmium (Cd) concentrations in fishery byproducts are an environmental concern, that might be reduced by enzymatic removal and adsorption of the contaminants during recycling the byproducts as animal food. We cloned the gene for Arthrobacter nicotinovorans serine protease (ANISEP), which was isolated from the hepatopancreas of the Japanese scallop (Patiopecten yessoensis) and has been found to be an effective enzyme for Cd(II) removal. The gene is 993 bp in length and encodes 330 amino acids, including the pre (1-30) and pro (31-111) sequences. The catalytic triad consists of His, Asp, and Ser. Sequence similarities indicate that ANISEP is a extracellular serine protease. X-ray crystallography revealed structural similarities between ANISEP and the trypsin-like serine protease NAALP from Nesterenkonia sp. Site-directed mutagenesis identified Ser171 as catalytic residue. The keratinolytic activity of ANISEP was 10-fold greater than that of trypsin. ANISEP digested Cd(II)-bound recombinant metallothionein MT-10a from Laternula elliptica, but did not release Cd. These results further suggest ANISEP is a trypsin-like serine protease that can release Cd from the Japanese scallop hepatopancreas because of its strong keratinolytic activity.
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21. |
Shearer AG,
Altman T,
Rhee CD,
( 2014 ) Finding sequences for over 270 orphan enzymes. PMID : 24826896 : DOI : 10.1371/journal.pone.0097250 PMC : PMC4020792 Abstract >>
Despite advances in sequencing technology, there are still significant numbers of well-characterized enzymatic activities for which there are no known associated sequences. These 'orphan enzymes' represent glaring holes in our biological understanding, and it is a top priority to reunite them with their coding sequences. Here we report a methodology for resolving orphan enzymes through a combination of database search and literature review. Using this method we were able to reconnect over 270 orphan enzymes with their corresponding sequence. This success points toward how we can systematically eliminate the remaining orphan enzymes and prevent the introduction of future orphan enzymes.
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22. |
Mihasan M,
Brandsch R,
( 2013 ) pAO1 of Arthrobacter nicotinovorans and the spread of catabolic traits by horizontal gene transfer in gram-positive soil bacteria. PMID : 23884627 : DOI : 10.1007/s00239-013-9576-x Abstract >>
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.
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23. |
( 1997 ) IS1473, a putative insertion sequence identified in the plasmid pAO1 from Arthrobacter nicotinovorans: isolation, characterization, and distribution among Arthrobacter species. PMID : 9073580 : DOI : 10.1006/plas.1996.1272 Abstract >>
A putative insertion sequence (IS1473) has been cloned and sequenced. The 1087-bp element was found between the moaA and the ndhA genes in the upstream region of the nicotine dehydrogenase (ndh) operon in the 160-kb pAO1 plasmid of Arthrobacter nicotinovorans. It is flanked by an imperfect repeat of 33 bp and carries two overlapping open reading frames which, by programmed -1 translational frameshifting, may produce a transposase of 36.735 Da with a pI = 10. 18. The deduced protein is similar to the transposases IS481 and IS1002 from Bordetella and IS476 from Xanthomonas campestris, all members of the IS3 family. The putative insertion element was found as a single copy in the pAO1 plasmid and absent on the chromosome of the A. nicotinovorans genome. Similar sequences were detected by hybridization on total DNA from Arthrobacter globiformis, Arthrobacter ramosus, and Arthrobacter ureafaciens.
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24. |
( 1996 ) MoaA of Arthrobacter nicotinovorans pAO1 involved in Mo-pterin cofactor synthesis is an Fe-S protein. PMID : 8706892 : DOI : 10.1016/0014-5793(96)00712-0 Abstract >>
MoaA, involved in an early step in the biosynthesis of the molybdopterin cofactor (MoCo), has not yet been characterized biochemically and the reaction it catalyzes is unknown. We overexpressed MoaA from pAO1 of Arthrobacter nicotinovorans in Escherichia coli as a N-terminal fusion with either glutathione-S-transferase or a 6-histidine tag. The pAO1 encoded MoaA as well as the fusion proteins functionally complement E. coli moaA mutants. Here we show that purified MoaA contains approximately 4 microM Fe and approximately 3 microM acid-labile S/microM protein. EPR spectroscopy revealed a predominant signal at g(av) = 2.01, indicative of a [3Fe-xS] cluster.
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25. |
( 1995 ) A pAO1-encoded molybdopterin cofactor gene (moaA) of Arthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein. PMID : 8588735 : Abstract >>
A gene homologous to moaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor of Escherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encoded moaA gene from A. nicotinovorans was expressed in E. coli as an active protein that functionally complemented moaA mutants. Its deduced amino acid sequence shows 43% identity to the E. coli MoaA, 44% to the NarAB gene product from Bacillus subtilis, and 42% to the gene product of two contiguous ORFs from Methanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in the fixZ gene product from Rhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement the moaA mutation in E. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy of moaA may not be unique in the A. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.
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26. |
Grether-Beck S,
Igloi GL,
Pust S,
Schilz E,
Decker K,
Brandsch R,
( 1994 ) Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans. PMID : 7815950 : DOI : 10.1111/j.1365-2958.1994.tb00484.x Abstract >>
The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M(r) 30,011, 14,924 and 87,677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hino genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.
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27. |
Gherna RL,
Richardson SH,
Rittenberg SC,
( 1965 ) The bacterial oxidation of nicotine. VI. The metabolism of 2,6-dihydroxypseudooxynicotine. PMID : 5835946 : Abstract >>
N/A
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28. |
Brandsch R,
Hinkkanen AE,
Mauch L,
Nagursky H,
Decker K,
( 1987 ) 6-Hydroxy-D-nicotine oxidase of Arthrobacter oxidans. Gene structure of the flavoenzyme and its relationship to 6-hydroxy-L-nicotine oxidase. PMID : 3622516 : DOI : 10.1111/j.1432-1033.1987.tb13338.x Abstract >>
The nucleotide sequence of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene of Arthrobacter oxidans is presented. This covalently flavinylated enzyme specifically oxidizes 6-hydroxy-D-nicotine to 6-hydroxy-N-methylmyosmine. Coinduced in the presence of nicotine is a 6-hydroxy-L-nicotine-specific enzyme, 6-hydroxy-L-nicotine oxidase (6-HLNO), with FAD noncovalently bound to the apoprotein. A comparison of the nucleotide-derived amino acid sequence of the 6-HDNO with the amino acid sequence data obtained from the purified 6-HLNO polypeptide suggests that the two enantiozymes expressed within the same cell are genetically unrelated. This conclusion is supported by the finding that the FAD-binding sites of the two enzymes are different. 6-HLNO exhibits at the amino-terminus of the polypeptide chain a dinucleotide-binding site characteristic for many other FAD- and NAD(P)-dependent enzymes. No such sequence was found in the nucleotide-derived amino acid sequence of 6-HDNO.
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29. |
( 2013 ) Evidence of a plasmid-encoded oxidative xylose-catabolic pathway in Arthrobacter nicotinovorans pAO1. PMID : 23063486 : DOI : 10.1016/j.resmic.2012.10.003 Abstract >>
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.
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30. |
( 1999 ) Horizontal gene transfer involved in the convergent evolution of the plasmid-encoded enantioselective 6-hydroxynicotine oxidases. PMID : 9929386 : Abstract >>
The D- and L-specific nicotine oxidases are flavoproteins involved in the oxidative degradation of nicotine by the Gram-positive soil bacterium Arthrobacter nicotinovorans. Their structural genes are located on a 160-kbp plasmid together with those of other nicotine-degrading enzymes. They are structurally unrelated at the DNA as well as at the protein level. Each of these oxidases possesses a high degree of substrate specificity; their catalytic stereoselectivity is absolute, although they are able to bind both enantiomeric substrates with a similar affinity. It appears that the existence of these enzymes is the result of convergent evolution. The amino acid sequence of 6-hydroxy-l-nicotine oxidase (EC 1.5.3.6) as derived from the respective structural gene shows considerable structural similarity with eukaryotic monoamine oxidases (EC 1.4.3.4) but not with monoamine oxidases from prokaryotic bacteria including those of the genus Arthrobacter. These similarities are not confined to the nucleotide-binding sites. A 100-amino acid stretch at the N-terminal regions of 6-hydroxy-l-nicotine oxidase and human monoamine oxidases A possess a 35% homology. Overall, 27.0, 26.9, and 25.8% of the amino acid positions of the monoamine oxidases of Aspergillus niger (N), humans (A), and rainbow trout (Salmo gairdneri) are identical to those of 6-hydroxy-l-nicotine oxidase (Smith-Waterman algorithm). In addition, the G+C content of the latter enzyme is in the range of that of eukaryotic monoamine oxidases and definitely lower than that of the A. nicotinovorans DNA and even that of the pAO1 DNA. The primary structure of 6-hydroxy-d-nicotine oxidase (EC 1.5.3.5) does not reveal its evolutionary history as easily. Significant similarities are found with a mitomycin radical oxidase from Streptomyces lavendulae (23.3%) and a "hypothetical protein" from Mycobacterium tuberculosis (26.0%). It is proposed that the plasmid-encoded gene of 6-hydroxy-l-nicotine oxidase evolved after horizontal transfer from an eukaryotic source.
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31. |
( 1998 ) Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans. PMID : 9878353 : DOI : 10.1006/jmbi.1998.2227 Abstract >>
Arthrobacter nicotinovorans is a Gram-positive aerobic soil bacterium able to grow on nicotine as its sole source of carbon and nitrogen. The initial steps of nicotine catabolism are catalyzed by nicotine dehydrogenase, the l- and d-specific 6-hydroxynicotine oxidases, and ketone dehydrogenase. The genes encoding these enzymes reside on a 160 kb plasmid, pAO1. The cccDNA of this plasmid was isolated in high purity and reasonable yield. It served as template material for the construction of a lambda-phage DNA library of the plasmid. The genes coding for 6-hydroxy-l-nicotine oxidase and for the subunits of the heterotrimeric ketone dehydrogenase were identified, subcloned and sequenced. The 6-hlno gene was identified as a 1278 bp open reading frame; its regulatory elements were also recognized. The derived primary structure of the monomer of apo-6-hydroxy-l-nicotine oxidase (46,264.5 Da) agrees with the data obtained by partial amino acid sequencing. 6-Hydroxy-l-nicotine oxidase and 6-hydroxy-d-nicotine oxidase were expressed in Escherichia coli and obtained in a state of high purity and crystallized. Ketone dehydrogenase (KDH) was found to be a heterotrimer with subunits of molecular mass 89,021.71, 26,778.65 and 17,638.88. The genes of KDH-A and KDH-B are juxtaposed; the A of the stop codon of KDH-A is used in the start codon of KDH-B, eliciting a frame shift. KDH-C is separated from KDH-A by 281 bp.
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32. |
( 1997 ) Molybdate-uptake genes and molybdopterin-biosynthesis genes on a bacterial plasmid--characterization of MoeA as a filament-forming protein with adenosinetriphosphatase activity. PMID : 9428706 : DOI : 10.1111/j.1432-1033.1997.0524a.x Abstract >>
A gene cluster consisting of homologs to Escherichia coli moaA, moeA, moaC and moaE, which encode enzymes involved in the biosynthesis of molybdopterin cofactor (MoCo), and to modA, modB and modC, which encode a high-affinity molybdate transporter, were identified on pAO1 of Arthrobacter nicotinovorans near genes of molybdopterin-dependent enzymes involved in nicotine degradation. This gene arrangement suggests a coordinated expression of the MoCo-dependent and the MoCo-biosynthesis genes and shows that catabolic plasmids may carry the transport and biosynthetic machinery for the synthesis of the cofactors needed for the functioning of the enzymes they encode. pAO1 MoeA functionally complemented E. coli moeA mutants. The overexpressed and purified protein, of molecular mass 44,500 Da, associated into high-molecular-mass complexes and spontaneously formed gels at concentrations above 1 mg/ml. Transmission electron microscopy and atomic force microscopy revealed that MoeA forms fibrilar structures. In the presence of Mg2+ MoeA exhibited ATPase activity (0.020 pmol ATP x pmol protein(-1) x min(-1)). ATP, ADP or AMP induced the disassembly of the MoeA fibers into aggregates. pAO1 MoeA shows 39% identity to the C-terminal domain of the rat neuroprotein gephyrin. Like gephyrin it binds to neurotubulin, but binds with preference to tubulin dimers.
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33. |
( 1997 ) Molecular cloning of levan fructotransferase gene from Arthrobacter nicotinovorans GS-9 and its expression in Escherichia coli. PMID : 9438987 : DOI : 10.1271/bbb.61.2076 Abstract >>
The gene encoding an extracellular levan fructotransferase, designated the lft gene, was cloned from the genomic DNA of Arthrobacter nicotinovorans GS-9, and expressed in Escherichia coli. It was found that a single open reading frame consisted of 1554 base pairs that encoded a polypeptide composed of a signal peptide of 33 amino acids and a mature protein of 484 amino acids (M(r) 53,152), and it was also found that a putative ribosome-binding site was present in the upstream from the ORF. The primary structure had no significant similarity with those of inulin fructotransferases, but had low similarity to the catalytic regions of other fructosylhydrolases. The expression of the lft gene was increased on a plasmid, pLFT-BB1, in which the lft gene was fused with alpha-peptide of the lacZ gene of pUC18. An E. coli transformant carrying pLFT-BB1 expressed six times as much activity of levan fructotransferase as that of the original strain, A. nicotinovorans GS-9.
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