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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A functional analysis of the Arthrobacter oxidans 6-hydroxy-D-nicotine oxidase (6-HDNO) gene promoter (-35 region TTGACA and -10 region TATCAAT) and the UUG translation start codon was performed using site-directed mutagenesis. Deletion of the C residue from the -10 promoter region or mutations introduced upstream of the -10 region resulted in an increased 6-HDNO expression in Escherichia coli cells in vivo and in both E. coli and A. oxidans coupled transcription-translation systems in vitro. From the identical behaviour of 6-HDNO promoter mutants in the heterologous and homologous systems, it is concluded that A. oxidans harbours an RNA polymerase functionally homologous to the E. coli sigma 70 and Bacillus subtilis sigma 43 polymerases. Replacement of the TTG codon (UUG translation initiation codon) with ATG led to a 3.7-fold increase in 6-HDNO expression in E. coli. This effect was less pronounced at higher promoter strengths, from 3.7 in the case of the 6-HDNO wild-type promoter, to 2.5 in the case of the consensus -10 region and to 1.7 in the case of the tac promoter. A double point mutation introduced close to the ribosome binding site resulted in almost the same increase in 6-HDNO expression (3.1-fold) as the TTG-to-ATG exchange. The failure of cAMP to stimulate 6-HDNO expression in the A. oxidans system indicated that expression of this gene in stationary phase cells is not regulated by cAMP-catabolite repressore protein-mediated mechanism of catabolite repression.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Gen Genet 1990 May
PMID:Functional analysis of the 5' regulatory region and the UUG translation initiation codon of the Arthrobacter oxidans 6-hydroxy-D-nicotine oxidase gene. 238 22

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.
Mol Gen Genet 1986 Jan
PMID:Plasmid pAO1 of Arthrobacter oxidans encodes 6-hydroxy-D-nicotine oxidase: cloning and expression of the gene in Escherichia coli. 300 41

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.
J Mol Evol 1999 Feb
PMID:Horizontal gene transfer involved in the convergent evolution of the plasmid-encoded enantioselective 6-hydroxynicotine oxidases. 992 86

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.
J Mol Biol 2005 Sep 16
PMID:Crystal structure of 6-hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans. 1609 22

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.
J Mol Biol 2010 Feb 26
PMID:Crystal structure analysis of free and substrate-bound 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans. 2000 20