flavin-adenine-dinucleotide and methylamine

Nicotine is a natural alkaloid produced by tobacco plants, and the mechanisms of its catabolism by microorganisms are diverse. In the present study, we reported the mutation, cloning, and identification of two novel genes involved in nicotine degradation from the newly isolated Pseudomonas sp. strain HZN6. Transposon mutagenesis identified a HZN6 mutant in which the nicotine-degrading pathway was blocked at pseudooxynicotine. A 3,874-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR. Two open reading frames (designated pao and sap) were analyzed, and the deduced amino acid sequences shared 29% identity with 6-hydroxy-l-nicotine oxidase from Arthrobacter nicotinovorans and 49% identity with an aldehyde dehydrogenase from Bartonella henselae. Both pao and sap were cloned and functionally expressed in recombinant Escherichia coli BL21. The pao gene encoded a novel pseudooxynicotine amine oxidase with noncovalently bound flavin adenine dinucleotide (FAD) and exhibited substrate specificity removing the methylamine from pseudooxynicotine with the formation of 3-succinoylsemialdehyde-pyridine and hydrogen dioxide. The sap gene encoded a NADP(+)-dependent 3-succinoylsemialdehyde-pyridine dehydrogenase that catalyzed the dehydrogenation of 3-succinoylsemialdehyde-pyridine to 3-succinoyl-pyridine. Genetic analyses indicated that the pao gene played an essential role in nicotine or pseudooxynicotine mineralization in strain HZN6, whereas the sap gene did not. This study provides novel insight into the nicotine-degrading mechanism at the genetic level in Pseudomonas spp.

Topics: Bacterial Proteins; Butanones; Cloning, Molecular; Escherichia coli; Flavin-Adenine Dinucleotide; Genes, Bacterial; Methylamines; Molecular Sequence Data; Monoamine Oxidase; Nicotine; Pseudomonas; Pyridines; Succinate-Semialdehyde Dehydrogenase (NADP+)

Glycine decarboxylase consists of four protein components. Its structural and mechanistic heart is provided by the lipoic acid-containing H-protein which undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. Lipoic acid attached to a specific lysine side chain is assumed to act as a 'swinging arm' conveying the reactive dithiolane ring from one catalytic centre to another. The X-ray crystal structures of two forms of the H-protein have been determined. The lipoate cofactor is located in the loop of a hairpin configuration but following methylamine transfer it is pivoted to bind into a cleft at the surface of the H-protein. The lipoamide-methylamine arm is, therefore, not free to move in aqueous solvent.

Topics: Amino Acid Oxidoreductases; Amino Acid Sequence; Animals; Bacterial Proteins; Carrier Proteins; Cattle; Chickens; Computer Simulation; Crystallization; Crystallography, X-Ray; Flavin-Adenine Dinucleotide; Glycine Decarboxylase Complex; Glycine Decarboxylase Complex H-Protein; Glycine Dehydrogenase (Decarboxylating); Humans; Methylamines; Models, Molecular; Molecular Sequence Data; Motion; Mutagenesis, Site-Directed; Plant Proteins; Protein Conformation; Sequence Alignment; Sequence Homology, Amino Acid; Solvents; Thioctic Acid