Covalent flavinylation of 6-hydroxy-D-nicotine oxidase involves an energy-requiring process

AbstractE. coli cells harbouring the recombinant plasmid pDB222 with the 6-HDNO gene under the control of the tac-promotor were induced with IPTG to synthesize a high amount of 6-HDNO protein. Part of this protein was present as 6-HDNO apoenzyme. The proportion of 6-HDNO apoenzyme formed could be increased when the induction of 6-HDNO synthesis by IPTG was performed in the presence of the inhibitor diphenyleneiodonium. The 6-HDNO apoenzyme thus formed could be transformed into enzymatically active holoenzyme in the presence of FAD by a process requiring an energy-generating system consisting of ATP, phosphoenolpyruvate and pyruvate kinase. This finding suggests that an enzymatic step(s) is (are) involved in the covalent flavinylation of 6-HDNO

Expression and flavinylation of Arthrobacter oxydans 6-hydroxy-D-nicotine oxidase in Bacillus subtilis

6-Hydroxy-d-nicotine oxidase (6-HDNO) of Arthrobacter oxydans, an enzyme inducible by dl-nicotine, contains FAD covalently bound via an 8α-N(3)His linkage. Expression of the gene encoding 6-HDNO and flavinylation of the protein were studied in Bacillus subtilis. In this heterologous system the following findings were made. 1. An enzymically active covalently flavinylated 6-HDNO of normal size can be expressed in B. subtilis. 2. The natural promoter of the 6-HDNO gene appeared inefficient in B. subtilis. The B. subtilis sdh promoter, when inserted upstream of the A. oxydans promoter, increased 6-HDNO expression >50-fold. 3. Expression of the 6-HDNO gene from plasmids in B. subtilis was, independently of the promoter construct used, stimulated more than fivefold by dl-nicotine in the growth medium. It is concluded that flavinylation of 6-HDNO is possibly autocatalytic and mediated by factors generally found in bacterial cells

Sequence of the 165-Kilobase Catabolic Plasmid pAO1 from Arthrobacter nicotinovorans and Identification of a pAO1-Dependent Nicotine Uptake System

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 μM. 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

Cloning and purification of a tetrameric oxidoreductase from Arthrobacter nicotinovorans pAO1

The pAO1 megaplasmid of Arthrobacter nicotinovorans encodes two different pathways: one for nicotine metabolism and an putative sugar catabolic pathway. One open reading frame, orf40, from the latter pathway was cloned, purified to homogenity and partially characterized. It consist of an tetrameric oxidoreductase containing atoms of zinc per molecule of monomer. A possible role in the metabolism of Arthrobacter nicotinovorans is postulated

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

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

CLONING AND PURIFICATION OFA REPRESSOR PROTEIN FROM ARTHROBACTER NICOTINOVORANS PAO1

The pAO1 megaplasmid of A. nicotinovorans consists of 165 ORF's related mainly to nicotine degradation, uptake and utilization of carbohydrates, amino acids and sarcosine The putative sugar catabolic pathway consists of 11 ORFś organized as a single operon and coding for an ABC-type sugar-transport system and several putative oxidoreductases and dehydrogenases. The current work is focused on orf32, a putative PdhR related protein, most probably involved in the control of the whole operon. The approx. 700 kb orf32 gene was cloned in the pH6EX3 plasmid vector and the gene product purified to homogeneity as a 29 kDa His-tagged recombinant protein. As indicated by GPC, it consists of a monomeric protein with a native molecular weight of 32 kDa. The specific UV/Vis spectra showed only a single peak at 280 nm common for all proteins and did not indicated the presence of any colored cofactors. This is in good agreement with the fact that PdhR-family proteins contain a winged helix-turn-helix (wHTH) domain responsible for DNA binding, and not a Zn-finger or any other metal containing domain

Plasmids for Nicotine-Dependent and -Independent Gene Expression in Arthrobacter nicotinovorans and Other Arthrobacter Species

The first inducible Arthrobacter overexpression system, based on the promoter/operator and the repressor of the 6-d-hydroxynicotine oxidase gene of Arthrobacter nicotinovorans, is described here. Nicotine-dependent overproduction and affinity purification of recombinant proteins are presented. The system will allow the production of complex enzymes and genetic complementation studies in Arthrobacter species

Characterization of PmfR, the Transcriptional Activator of the pAO1-Borne purU-mabO-folD Operon of Arthrobacter nicotinovorans

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 γ-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 γ-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