Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans

Abstract

Arthrobacter nicotinovorans is a Gram-positive aerobic soil bacterium able to grow on nicotine as its sole source of carbon and nitrogen. The initial steps of nicotine catabolism are catalyzed by nicotine dehydrogenase, the l- and d-specific 6-hydroxynicotine oxidases, and ketone dehydrogenase. The genes encoding these enzymes reside on a 160 kb plasmid, pAO1. The cccDNA of this plasmid was isolated in high purity and reasonable yield. It served as template material for the construction of a λ-phage DNA library of the plasmid. The genes coding for 6-hydroxy-l-nicotine oxidase and for the subunits of the heterotrimeric ketone dehydrogenase were identified, subcloned and sequenced. The 6-hlno gene was identified as a 1278 bp open reading frame; its regulatory elements were also recognized. The derived primary structure of the monomer of apo-6-hydroxy-l-nicotine oxidase (46,264.5 Da) agrees with the data obtained by partial amino acid sequencing. 6-Hydroxy-l-nicotine oxidase and 6-hydroxy-d-nicotine oxidase were expressed in Escherichia coli and obtained in a state of high purity and crystallized. Ketone dehydrogenase (KDH) was found to be a heterotrimer with subunits of molecular mass 89,021.71, 26,778.65 and 17,638.88. The genes of KDH-A and KDH-B are juxtaposed; the A of the stop codon of KDH-A is used in the start codon of KDH-B, eliciting a frame shift. KDH-C is separated from KDH-A by 281 bp.