Nicotine dehydrogenase from Arthrobacter oxidans: A molybdenum-containing hydroxylase

Abstract The nicotine dehydrogenase from Arthrobacter oxidans was purified 40-fold to homogeneity with 26% recovery. SDS-polyacrylamide gel electrophoresis of the enzyme revealed three protein bands corresponding to M _r of 82 000, 30 000 and 15 000. The M _r of the native enzyme was calculated to be 12 0000 by gel chromatography. The enzyme contained about 1 FAD, 1 molybdenum, 4 iron and 2 labile sulfur.     

Plasmid-mediated nicotine degradation inArthrobacter oxidans

The spontaneous loss byArthrobacter oxidans cells of the nicotine-degrading ability (Nic⁺) was 0.06%. It could be increased by treatment with plasmid-curing agents up to 8%. It was possible by conjugation to restore the Nic⁺ phenotype in such cured derivatives and to transfer the Nic⁺ character to Nic^- Arthrobacter species. Plasmid DNA, 160 kb in size as judged by contour length measurements, could be isolated from cleared lysates ofA. oxidans cells by acridine yellow chromatography. Agarose gel electrophoresis of DNA isolated fromArthrobacter exconjugates revealed the occurrence of plasmid DNA within these strains; its mobility was similar to that of the plasmid DNA present inA. oxidans. Although the expression and inducibility of the transferred genes was poor in most of theArthrobacter species exconjugants, apparently authentic 6-hydroxy-l-nicotine oxidase could be identified in these cells after enrichment by an enzyme-specific chromatography.Abbreviations 6-HDNO 6-hydroxy-d-nicotine oxidase - 6-HLNO 6-hydroxy-l-nicotine oxidase - kb kilobase - Nic⁺ ability to usel- ord-nicotine as sole carbon and nitrogen source - Nic^- absence of Nic⁺ character Enzymes (EC 1.5.3.5.) 6-Hydroxy-l-nicotine oxidase, 6-hydroxy-l-nicotine: oxygen oxidoreductase - (EC 1.5.3.6.) 6-hydroxy-d-nicotine oxidase, 6-hydroxy-d-nicotine: oxygen oxidoreductase - (EC 3.1.4.22) ribonuclease A, ribonucleate 3-pyrimidino-oligo-nucleotidohydrolase     

Isolation and partial characterization of plasmid DNA from Arthrobacter oxidans

A method for the extraction of the high molecular weight plasmid AO 1 from the gram-positive soil bacterium Arthrobacter oxidans is presented.Following digestion of this DNA with the restriction endonucleases Accl, Bam HI, Eco RI and Hind III, an average molecular mass of 157.8 kb was estimated. This value is in good agreement with the 160 kb size determined previously by electron microscopy (Brandsch et al. 1982).Using the same method, no plasmid DNA was found in strains of the genus Arthrobacter which do not degrade nicotine, e.g., A. albidus, A. globiformis and A. auricans.Abbreviations EDTA ethylenediaminetetraacetic acid - Kb kilobasepairs - SDS sodium dodecyl sulfate - Tris Tris-(hydroxymethyl)-aminomethan     

Covalently bound FAD in D-6-hydroxynicotine oxidase from Arthrobacter oxidans

In Arthrobacter oxidans, an enzymatically inactive protein which is in a close biosynthetic relationship to the active D-6-hydroxynicotine oxidase and may serve as a precursor for its formation was purified and shown to be homogeneous by gel electrophoresis. It consists of one polypeptide chain of about the same molecular weight (50 000 daltons) as the active enzyme. The purified protein lacks the absorption in the visible range characteristic of flavoproteins. Amino acid analysis and peptide mapping yielded similar results for both proteins. They also share the same C-terminal amino acids,-lysinetyrosine; the N-terminal residue is serine in the case of D-6-hydroxynicotine oxidase while that of the coinduced protein was found to be blocked.     

Induction of d-6-hydroxynicotine oxidase in resting cells of Arthrobacter oxidans

Resting cell suspensions of Arthrobacter oxidans were shown to synthesize the inducible enantiozyme, d-6-hydroxynicotine oxidase, in the presence of d-nicotine or d-6-hydroxynicotine. The corresponding l-enantiomers, as well as -methylaminopropyl-(6-OH-pyridyl-3)-ketone, which is the product of the reaction catalyzed by the enzyme, were ineffective as inducers. l-6-Hydroxynicotine inhibited induction by d-nicotine and d-6-hydroxynicotine while l-nicotine inhibited induction by d-6-hydroxynicotine and had no effect on induction by d-nicotine. Enzyme induction was also found to be inhibited by glucose, 2-deoxy-d-glucose and by several intermediates of the tricarboxylic acid cycle. An absolute requirement for protein synthesis and for oxygen was also demonstrated to be necessary for the reactions involved in the covalent attachment of flavin adenine dinucleotide to pre-existing precursor protein to yield the catalytically active d-6-hydroxynicotine oxidase.H. C. R. completed these studies while on sabbatical leave from the Department of Botany and Microbiology, Arizona State University, Tempe, Arizona 85281, U.S.A.     

6-Hydroxy-D-nicotine oxidase of Arthrobacter oxidans. Gene structure of the flavoenzyme and its relationship to 6-hydroxy-L-nicotine oxidase

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.     

Localization of the enantiozymes of 6-hydroxy-nicotine oxidase in Arthrobacter oxidans by electron immunochemistry.

During the course of growth of Arthrobacter oxidans, induction of the enantiozymes 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase occurred in the presence of DL-nicotine. Cryoultramicrotomed sections obtained from cells grown to stationary phase were gold immunolabeled. The results obtained demonstrate that both enzymes are localized in the cytoplasm.     

The structure of extracellular polysaccharide of Arthrobacter globiformis

An extracellular polysaccharide from Arthrobacter globiformis is composed of N-acetyl-D-glucosamine, N-acetyl-D-fucosamine, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid and O-acetyl groups in the ratio 1:1:2:1. On the basis of solvolysis with anhydrous hydrogen fluoride, which resulted in a tetrasaccharide fragment, and analysis by 1H and 13NMR spectroscopy, it was concluded that the polysaccharide has the following structure: (formula; see text).     

Crystal structure of 6-hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans

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.     

The effect of gyrase inhibitors and cyclic AMP on induction and glucose repression of the 6-hydroxy-nicotine oxidases in Arthrobacter oxidans

The induction by d,l-nicotine of the enantiozymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase in Archrobacter oxidans was differently affected by the inhibitors of Escherichia coli gyrase, novobiocin and nalidixic acid. These compounds inhibited 6-hydroxy-L-nicotine oxidase induction slightly, but led to an increase in the level of 6-hydroxy-D-nicotine oxidase activity. Furthermore, the specific repression by glucose of 6-hydroxy-D-nicotine oxidase synthesis was not abolished by the addition of cAMP but by that of novobiocin.Abbreviations 6-HDNO 6-hydroxy-D-nicotine oxidase - 6-HLNO 6-hydroxy-L-nicotine oxidase - cAMP cyclic 3,5-adenosine monophosphate - Enzymes Adenylate cyclase - ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1) - cAMP-phosphodiesterase 3:5-cyclic-nucleotide 5-nucleotido-hydrolase (EC 3.1.4.17) - DNA gyrase DNA topoisomerase II (EC 5.99) - DNA polymerase deoxynucleosidetriphosphate: DNA desoxynucleotidyl-transferase (EC 2.7.7.7) - 6-hydroxy-L-nicotine oxidase 6-hydroxy-L-nicotine: oxygen oxidoreductase (EC 1.5.3.5) - 6-hydroxy-D-nicotine oxidase 6-hydroxy-D-nicotine: oxygen oxidoreductase (EC 1.5.3.6) - -lactamase penicillin amido--lactamhydrolase (EC 3.5.2.6) - nicotine dehydrogenase nicotine: (acceptor)6-oxidoreductase (hydroxylating) (EC 1.5.99.4)