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

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 o f this plasmid was isolated in high purity and reasonable yield. It served as template material for the construction of a h-phage DNA library of the p lasmid. The genes coding for 6-hydroxy-L-nicotine oxidase and for the subun its of the heterotrimeric ketone dehydrogenase were identified, subcloned a nd sequenced. The 6-hlno gene was identified as a 1278 by open reading fram e; its regulatory elements were also recognized. The derived primary struct ure 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-nicot ine oxidase and 6-hydroxy-D-nicotine oxidase were expressed in Escherichia coli and obtained in a state of high purity and crystallized. Ketone dehydr ogenase (KDH) was found to be a heterotrimer with subunits of molecular mas s 89,021.71, 26,778.65 and 17,638.88. The genes of KDH-A and KDH-B are juxt aposed; 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. (C) 19 98 Academic Press.