University of Groningen a Novel Gamma-N-Methylaminobutyrate
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University of Groningen A novel gamma-N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1 Chiribau, CB; Sandu, C; Fraaije, M; Schiltz, E; Brandsch, R; Chiribau, Calin B. Published in: European Journal of Biochemistry DOI: 10.1111/j.1432-1033.2004.04432.x IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2004 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Chiribau, CB., Sandu, C., Fraaije, M., Schiltz, E., Brandsch, R., & Chiribau, C. B. (2004). A novel gamma- N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1. European Journal of Biochemistry, 271(23-24), 4677-4684. https://doi.org/10.1111/j.1432-1033.2004.04432.x Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-09-2021 Eur. J. Biochem. 271, 4677–4684 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04432.x A novel c-N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1 Calin B. Chiribau1, Cristinel Sandu1, Marco Fraaije2, Emile Schiltz3 and Roderich Brandsch1 1Institute of Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany; 2Laboratory of Biochemistry, University of Groningen, the Netherlands; 3Institute of Organic Chemistry and Biochemistry, University of Freiburg, Freiburg, Germany Nicotine catabolism, linked in Arthrobacter nicotinovorans of covalent attachment of FAD and confirmed Trp66 as to the presence of the megaplasmid pAO1, leads to the for- essential for FAD binding, for enzyme activity and for the mation of c-N-methylaminobutyrate from the pyrrolidine spectral properties of the wild-type enzyme. A Km of 140 lM )1 ring of the alkaloid. Until now the metabolic fate of and a kcat of 800 s was determined when c-N-methyl- c-N-methylaminobutyrate has been unknown. pAO1 carries aminobutyrate was used as the substrate. Sarcosine was also a cluster of ORFs with similarity to sarcosine and dimeth- turned over by the enzyme, but at a rate 200-fold slower than ylglycine dehydrogenases and oxidases, to the bifunctional c-N-methylaminobutyrate. This novel enzyme activity enzyme methylenetetrahydrofolate dehydrogenase/cyclo- revealed that the first step in channelling the c-N-methyl- hydrolase and to formyltetrahydrofolate deformylase. We aminobutyrate generated from nicotine into the cell meta- cloned and expressed the gene carrying the sarcosine dehy- bolism proceeds by its oxidative demethylation. drogenase-like ORF and showed, by enzyme activity, spec- Keywords: Arthrobacter nicotinovorans; c-N-methylamino- trophotometric methods and identification of the reaction butyrate oxidase; megaplasmid pAO1; nicotine degradation; product as c-aminobutyrate, that the predicted 89 395 Da sarcosine oxidase. flavoprotein is a demethylating c-N-methylaminobutyrate oxidase. Site-directed mutagenesis identified His67 as the site The bacterial soil community plays a pivotal role in the heterotrimeric enzyme of the xanthine dehydrogenase biodegradation of an almost unlimited spectrum of natural family, which carries a molybdenum cofactor (MoCo), a and man-made organic compounds, among them the FAD moiety and two iron-sulphur clusters [15,16]. Next, tobacco alkaloid nicotine. Perhaps analysed in greatest the pyrrolidine ring of 6-hydroxy-L-nicotine is oxidized by detail is the pathway of nicotine degradation as it takes 6-hydroxy-L-nicotine oxidase [17]. A second hydroxylation place in Arthrobacter nicotinovorans (formerly known as of the pyridine ring of nicotine is performed by ketone A. oxydans). Pioneering work on the identification of the dehydrogenase [18], an enzyme similar to nicotine enzymatic steps of this oxidative catabolic pathway was dehydrogenase, yielding 2,6-dihydoxypseudooxynicotine performed in the early 1960s by Karl Decker and [N-methylaminopropyl-(2,6-dihydroxypyridyl-3)-ketone] co-workers at the University of Freiburg, Germany [1–8], (Fig. 1). Cleavage of 2,6-dihydoxypseudooxynicotine by an and by Sidney C. Rittenberg and co-workers at the as yet unknown enzyme, results in the formation of 2, University of Southern California (Los Angeles, CA, USA) 6-dihydroxypyridine and c-N-methylaminobutyrate [6,14]. [9–14]. The first step in the breakdown of L-nicotine, the 2,6-Dihydroxypyridine is hydroxylated to 2,3,6-trihydroxy- natural product synthesized by the tobacco plant, is the pyridine by the FAD-dependent 2,6-dihydroxypyridine hydroxylation of the pyridine ring of nicotine in position hydroxylase [19] and, in the presence of O2, spontaneously six. This step is catalysed by nicotine dehydrogenase, a forms a blue pigment, known as nicotine blue. The metabolic fate of c-N-methylaminobutyrate was unknown until now. Correspondence to R. Brandsch, Institut fu¨ r Biochemie und Moleku- Biodegradation of nicotine by A. nicotinovorans is linked larbiologie, Hermann-Herder-Str. 7, 79104 Freiburg, Germany. to the presence of the megaplasmid, pAO1 [20]. The recent Fax: +49 761 2035253, Tel.: +49 761 2035231, elucidation of the DNA sequence of pAO1 revealed the E-mail: [email protected] modular organization of the enzyme genes involved in Abbreviations:MABO,c-N-methylaminobutyrate oxidase; MoCo, nicotine degradation [21]. Next to a nic-gene cluster [19], molybdenum cofactor. there is a cluster of genes on pAO1 encoding the complete Note: this article was dedicated to Karl Decker for the occasion of his enzymatic pathway responsible for the synthesis of MoCo, 80th birthday. required for enzyme activity by nicotine dehydrogenase and (Received 2 September 2004, revised 7 October 2004, ketone dehydrogenase, and a gene cluster of an ABC accepted 13 October 2004) molybdenum transporter. Adjacent to the nic-gene cluster is 4678 C. B. Chiribau et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 1. Breakdown of nicotine by Arthro- bacter nicotinovorans pAO1 (see the text for details). 6HLNO, 6-hydroxy-L-nicotine oxi- dase; KDH, ketone dehydrogenase; MABO, c-N-methylaminobutyrate oxidase; NDH, nicotine dehydrogenase. a set of hypothetical genes encoding a predicted flavo- Purification of MABO enzyme similar to mitochondrial and bacterial sarcosine and dimethylglycine dehydrogenases and oxidases (ORF63), The recombinant plasmid carrying the MABO gene was and two putative enzymes of tetrahydrofolate metabolism transformed into E. coli BL21 (Novagen, Schwalbach, (ORF64 and ORF62) [21]. Germany)andselectedon50lgÆmL)1 of ampicillin. One- In the present work we show that the protein encoded by hundred millilitres of LB medium was inoculated with a the sarcosine dehydrogenase-like ORF63 represents a novel single colony, cultured overnight at 30 °Candusedto enzyme, specific for the oxidative demethylation of inoculate 1 L of LB medium. MABO overexpression was c-N-methylaminobutyrate generated from 2,6-dihydroxy- induced with 0.3 mM isopropyl thio-b-D-galactoside at pseudooxynicotine. Identification of this enzyme extends 22 °C for 24 h. Bacteria were harvested at 5000 g,resus- our knowledge about the catabolic pathways of nicotine in pended in 40 mM Hepes buffer, pH 7.4, containing 0.5 M bacteria and demonstrates that the first step in the metabolic NaCl, and disrupted with the aid of a Branson sonifier. turnover of c-N-methylaminobutyrate consists of its deme- The supernatant obtained by centrifugation of the bacterial thylation. lysate at 13 000 g was used to isolate the proteins on Ni-chelating Sepharose, as described by the supplier of the Experimental procedures Sepharose (Amersham Biosciences, Freiburg, Germany). The isolated protein was analysed by SDS/PAGE on 10% (w/v) polyacrylamide gels. Superdex S-200 permeation Bacterial strains and growth conditions chromatography, for determining the size of the native A. nicotinovorans pAO1 was grown at 30 °Concitrate protein, was performed with the aid of a Mini-Maxi Ready medium supplemented with vitamins, trace elements [22] Rack device, according to the suggestions of the supplier and 5 mM of L-nicotine, as required. Growth of the (Amersham Biosciences). culture was monitored by the increase in absorption at 600 nm. Escherichia coli XL1-Blue was employed as a Determination of enzyme activity host for plasmids and was cultured at 37 °ConLB (Luria–Bertani) medium, supplemented with the appro- Enzyme activity was determined by using the peroxidase- priate antibiotics. coupled assay, consisting of 20 mM potassium phosphate buffer, pH 10, 25 lM to 10 mM c-aminobutyrate or )1 1–100 mM sarcosine as substrates, 10 IUÆmL of horse- Cloning of the c-N-methylaminobutyrate oxidase radish peroxidase (Sigma, Steinheim,