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.     

Covalently bound flavin as prosthetic group of choline oxidase.

Highly purified choline oxidase of $\text{Arthrobacter globiformis}$ fluoresced as a yellow band on SDS gel in 7% acetic acid. The absorption spectrum of the enzyme showed marked hypsochromic shift of the second absorption band. Aminoacyl flavin obtained from this enzyme was identified with 8α-[N(3)-histidyl]FAD.     

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.     

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.     

13C, 15N, and 31P NMR studies on 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans

The interaction between the apoprotein of 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans and the prosthetic group FAD has been investigated by 13C, 15N, and 31P NMR techniques. The FAD prosthetic group was selectively enriched in 13C and 15N isotopes by adding isotopically labeled riboflavin derivatives to the growth medium of riboflavin-requiring mutant cells. In the oxidized state the chemical shift of the C(7) and C(8) atoms indicates that the xylene moiety of the isoalloxazine ring is embedded in a hydrophobic environment. The polarization of the isoalloxazine ring as a whole is, however, much more comparable to that of free flavin in a polar and protic environment than to free flavin in an apolar environment. The polarization of the ring system can be ascribed to strong hydrogen bonds between the apoprotein and the two carbonyl groups. The binding of the competitive inhibitor, 6-hydroxy-D-nicotine, influences the resonances of the C(4a) and the N(5) atoms strongly. It is suggested that these shifts are due to a strong hydrogen-bonding interaction between the N(5) atom and the inhibitor. On reduction all resonances, except those of the C(10a) and the N(1) atoms, shift upfield, indicating the increased electron density in the ring system. In the dithionite-reduced enzyme, the ring system is bent at the N(5) position. Due to the bending of the N(5) atom and the sp2 hybridized N(10) atom, electron density from the N(10) atom is reallocated at the C(4) carbonyl group. In contrast, in the substrate-reduced enzyme the N(5) atom is almost completely sp2 hybridized, yielding a rather planar isoalloxazine ring.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of arsenite oxidase from Arthrobacter sp.

The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE. The V _max and K _m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of the enzyme are integral part of the protein.     

Purification and characterization of choline oxidase from Arthrobacter globiformis

Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis) and an isoelectric point (pI) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline + O2 leads to betaine aldehyde + H2O2, betaine aldehyde + O2 + H2O leads to betaine + H2O2. The enzyme was highly specific for choline and betaine aldehyde (relative reaction velocities: choline, 100%; betaine aldehyde, 46%; N,N-dimethylaminoethanol, 5.2%; triethanolamine, 2.6%; diethanolamine, 0.8%; monoethanolamine, N-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5.     

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.     

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.     

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     

Identification of the covalently bound flavin of L-gulono-gamma-lactone oxidase.

A specific mRNA for a structural lipoprotein of the $\text{Escherichia}\text{coli}$ outer membrane was translated in a wheat germ cell-free protein synthesizing system, S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) had neither stimulative nor inhibitory effect on the translation. When the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two peaks appeared at the appearent molecular weights of about 15,000 and about 7,500. Both products were cross-reactive with antiserum against the lipoprotein.     

Purification and properties of synephrinase from Arthrobacter synephrinum

Synephrinase, an enzyme catalyzing the conversion of (-)-synephrine into p-hydroxyphenylacetaldehyde and methylamine, was purified to apparent homogeneity from the cell-free extracts of Arthrobacter synephrinum grown on (+/-)-synephrine as the sole source of carbon and nitrogen. A 40-fold purification was sufficient to produce synephrinase that is apparently homogeneous as judged by native polyacrylamide gel electrophoresis and has a specific activity of 1.8 mumol product formed/min/mg protein. Thus, the enzyme is a relatively abundant enzyme, perhaps comprising as much as 2.5% of the total protein. The enzyme essentially required a sulfhydryl compound for its activity. Metal ions like Mg2+, Ca2+, and Mn2+ stimulated the enzyme activity. Metal chelating agents, thiol reagents, denaturing agents, and metal ions like Zn2+, Hg2+, Ag1+, and Cu2+ inhibited synephrinase activity. Apart from (-)-synephrine, the enzyme acted upon (+/-)-octopamine and beta-methoxysynephrine. Molecular oxygen was not utilized during the course of the reaction. The molecular mass of the enzyme as determined by Sephadex G-200 chromatography, was around 156,000. The enzyme was made up of four identical subunits with a molecular mass of 42,000.     

Purification and properties of an endo-inulinase from an Arthrobacter sp.

Extracellular endo-inulinase of Arthrobacter sp. S37 was purified 63-fold, giving a single band on PAGE with activity staining. The Mr was estimated as 75 kDa by SDS-PAGE. The first 31 amino acids of the N-terminal sequence was determined. The endo-inulinase hydrolyzed inulin mainly into inulo-triose (F3), inulo-tetraose (F4) and inulo-pentaose (F5) optimally at pH 7.5 and 50°C. © Rapid Science Ltd. 1998