The structure of nicotine blue from Arthrobacter oxidans

Summary Nicotine blue was separated into two major components. Their chromophores are anions of the diazadiphenoquinones (1) (nicotine blue I) and (2) (nicotine blue II). The dihydrate of the potassium salt of (1) is the major component of the extracellular crystals which are excreted on a solid medium. The acid-labile nicotine blue II is water-soluble and the main coloring agent of the agar and culture broth respectively.     

Mechanistic aspects of the covalent flavoprotein dimethylglycine oxidase of Arthrobacter globiformis studied by stopped-flow spectrophotometry

Dimethylglycine oxidase (DMGO) is a covalent flavoenzyme from Arthrobacter globiformis that catalyzes the oxidative demethylation of dimethylglycine to yield sarcosine, formaldehyde, and hydrogen peroxide. Stopped-flow and steady-state kinetic studies have been used to study the reductive and oxidative half-reactions using dimethylglycine and O2 as substrates. The reductive half-reaction is triphasic. The rate of the fast phase is dependent on substrate concentration, involves flavin reduction, and has a limiting rate constant of 244 s(-1). This phase also displays a kinetic isotope effect of 2.9. Completion of the first kinetic phase generates an intermediate with broad spectral signature between 350 and 500 nm, which is attributed to a reduced enzyme-iminium charge-transfer species, similar to the purple intermediate that accumulates in reactions of D-amino acid oxidase (DAAO) with alanine. The second phase (16 s(-1)) is independent of substrate concentration and is attributed to iminium hydrolysis/deprotonation. The third phase (2 s(-1)) is attributed to product release, the rate of which is less than the steady-state turnover rate (10.6 s(-1)). Flavin oxidation of dithionite- and dimethylglycine-reduced enzyme by O2 occurs in a single phase, and the rate shows a linear dependence on oxygen concentration, giving bimolecular rate constants of 342 and 201 mM(-1) x s(-1), respectively. Enzyme-monitored turnover experiments indicate that decay of the reduced enzyme-iminium intermediate is rate-limiting, consistent with rate constants determined from single turnover studies. A minimal kinetic mechanism is presented, which establishes a close relationship to the mechanism of action of DAAO. The covalent flavin in dimethylglycine oxidase is identified as an alphaN1-histidyl48-FAD, and equilibrium titration studies establish a single redox center that displays typical flavoprotein 'oxidase' characteristics.     

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant.

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.     

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.     

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.     

Regulation of synthesis of alkaline phosphatase by deoxyribonucleic acid synthesis in a constitutive mutant of Bacillus subtilis

Hiraga, Sota (Osaka University, Osaka, Japan). Regulation of synthesis of alkaline phosphatase by deoxyribonucleic acid synthesis in a constitutive mutant of Bacillus subtilis. J. Bacteriol. 91:2192-2199. 1966.-It was found that synthesis of alkaline phosphatase (APase) correlated with deoxyribonucleic acid (DNA) synthesis in a partially constitutive mutant of Bacillus subtilis. When cultures of the mutant were made to undergo synchronous growth by germination of spores in an excess-phosphate medium, synthesis of APase was repressed at the beginning of DNA synthesis. If the initiation of DNA synthesis was inhibited by thymine starvation, the repression of APase was not observed. When DNA synthesis, previously initiated, was inhibited by thymine or uracil starvation, or by addition of mitomycin C, the repression was partially released at a later stage. In contrast, this correlation between repression and DNA synthesis was not observed in a repressible strain.     

Plasmid pAO1 of Arthrobacter oxidans encodes 6-hydroxy-D-nicotine oxidase: cloning and expression of the gene in Escherichia coli

Summary The 160 kb plasmid pAO1 from Arthrobacter oxidans (Brandsch and Decker 1984) was subcloned in Escherichia coli with the aid of the plasmid vectors pUR222 and pBR322. Screening of the recombinant clones for enzyme activity revealed that the flavoenzyme 6-hydroxy-d-nicotine oxidase (6-HDNO), one of the enzymes of the nicotine-degradative pathway in A. oxidans, is encoded on pAO1. Immunoprecipitation of ³⁵S-methionine-labelled E. coli cells with 6-HDNO-specific antiserum and expression of recombinant plasmid DNA in E. coli maxicells revealed that 6-HDNO is made as a 52,000 dalton protein, approximately 4,500 daltons larger than 6-HDNO from A. oxidans. The 6-HDNO activity was constitutively expressed in E. coli cells, possibly from an A. oxidans promoter, as shown by subcloning of the 6-HDNO gene in pBR322, using the expression vector pKK223-3 and the promoter probe vector pCB192.     

Purification and properties of neuraminidase isozymes in Arthrobacter ureafaciens mutant

An Arthrobacter ureafaciens mutant (M1057) capable of producing neuraminidase constitutively was isolated by NTG mutagenesis from A. ureafaciens KMS 3663. Four molecular species (L, M1, M2, and S) of neuraminidase isozymes were homogeneously purified from the mutant and parent strains by means of DEAE-cellulose, affinity chromatography, ammonium sulfate precipitation, chromatofocusing, and Ultrogel AcA44 gel filtration. The molecular weights of L, M1, M2, and S isozymes were shown to be approximately 88,000, 66,000, 66,000, and 52,000, respectively. The optimal pHs and Km values of these isozymes for N-acetylneuraminosyl-alpha,(2-6)-lactose were 4.5-5.5 and 0.6-0.8 mM. Neuraminidase L, M1, M2, and S were able to hydrolyze oligosaccharides, glycoproteins and gangliosides containing alpha,(2-3)-, alpha,(2-6)-, and alpha,(2-8)-linked N-acetylneuraminic acid. Among these isozymes isolated, isozyme S was most active on colominic acid.     

Regulation of protein synthesis by branched-chain amino acids in vivo

Recent advances in the understanding of mRNA translation have facilitated molecular studies on the regulation of protein synthesis by nutrients and the interplay between nutrients and hormonal signals. Numerous reports have established that, in skeletal muscle, the branched-chain amino acids (BCAAs) have the unique ability to initiate signal transduction pathways that modulate translation initiation. Of the BCAAs, leucine is the most potent. Oral administration of leucine to food-deprived rats enhances muscle protein synthesis, in part, through activation of the mRNA binding step of translation initiation. Interestingly, leucine signaling in skeletal muscle differs from that in liver, suggesting that the responses may be tissue specific. The purpose of this paper was to briefly review the current knowledge of how BCAAs act as regulators of protein synthesis in physiologically important tissues, with particular focus on the mechanisms by which BCAAs regulate translation initiation.     

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)     

Regulation of expression by divalent cations of a light-inducible gene in Arthrobacter photogonimos

Expression of the light-inducible (lipA) gene in Arthrobacter photogonimos is repressed by Ca²⁺ at a concentration greater than 0.1 M. Expression of lipA was induced by relatively high concentrations of Zn²⁺ Ni²⁺ or Co²⁺ in cell suspensions, an effect that was blocked by an increase in the concentration of Ca²⁺ in the medium. Zn²⁺ and other metals apparently overcame repression by Ca²⁺ by competing for a cellular binding site. Expression of lipA was also induced when the amount of free Ca²⁺ was lowered with ethylene-bis (oxyethylenenitrilo)tetraacetic acid (EGTA). Our results show that the lipA gene does not require Zn²⁺ or other divalent cation for expression and that it is regulated negatively by Ca²⁺.Accumulation of the mature product of this gene (light-inducible protein, LIP) was minimal in the presence of EGTA. Accumulation increased 10-to 20-fold when divalent cations such as Ca²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ were added to cell suspensions treated with chelator. These divalent cations, which allowed the protein to achieve a protease-resistant form on the cell surface, could be substituted by protease inhibitors such as antipain, leupeptin or 1,10-phenanthroline. Our data can be explained by a biparous mechanism in which divalent cations regulate both expression of the lipA gene and accumulation of the gene product.Abbreviations LIP light-inducible protein - BAPTA 1,2-bis(o-aminophenoxy)ethanc-N,N,N,N-tetraacetic acid