Molecular Deceleration Regulates Toxicant Release to Prevent Cell Damage in Pseudomonas putida S16 (DSM 28022).

The underlying molecular mechanisms of flavin-dependent amine oxidases remain relatively poorly understood, even though many of these enzymes have been reported. The nicotine oxidoreductase NicA2 is a crucial enzyme for the first step of nicotine degradation in Pseudomonas putida S16 (DSM 28022). Here, we present the crystal structure of a ternary complex comprising NicA2 residues 21 to 482, flavin adenine dinucleotide (FAD), and nicotine at 2.25 Å resolution. Unlike other, related structures, NicA2 does not have an associated diacyl glycerophospholipid, wraps its substrate more tightly, and has an intriguing exit passage in which nine bulky amino acid residues occlude the release of its toxic product, pseudooxynicotine (PN). The replacement of these bulky residues by amino acids with small side chains effectively increases the catalytic turnover rate of NicA2. Our results indicate that the passage in wild-type NicA2 effectively controls the rate of PN release and thus prevents its rapid intracellular accumulation. It gives ample time for PN to be converted to less-harmful substances by downstream enzymes such as pseudooxynicotine amine oxidase (Pnao) before its accumulation causes cell damage or even death. The temporal metabolic regulation mode revealed in this study may shed light on the production of cytotoxic compounds.IMPORTANCE Flavin-dependent amine oxidases have received extensive attention because of their importance in drug metabolism, Parkinson's disease, and neurotransmitter catabolism. However, the underlying molecular mechanisms remain relatively poorly understood. Here, combining the crystal structure of NicA2 (an enzyme in the first step of the bacterial nicotine degradation pathway in Pseudomonas putida S16 (DSM 28022)), biochemical analysis, and mutant construction, we found an intriguing exit passage in which bulky amino acid residues occlude the release of the toxic product of NicA2, in contrast to other, related structures. The selective product exportation register for NicA2 has proven to be beneficial to cell growth. Those seeking to produce cytotoxic compounds could greatly benefit from the use of such an export register mechanism.

Complete Genome Sequence of Sphingomonas sp. Strain NIC1, an Efficient Nicotine-Degrading Bacterium.

Sphingomonas sp. strain NIC1, an efficient nicotine-degrading bacterium, was isolated from tobacco leaves. Here, we present the complete genome sequence of strain NIC1, which contains one circular chromosome and two circular plasmids. The genomic information will provide insights into its molecular mechanism for nicotine degradation.

Molecular mechanism of nicotine degradation by a newly isolated strain, Ochrobactrum sp. strain SJY1.

A newly isolated strain, SJY1, identified as Ochrobactrum sp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designated vppB, vppD, and vppE, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. The vppB gene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-N-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-l-nicotine oxidase, 24%) from the pyridine pathway. The vppD gene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) from Pseudomonas putida strain S16, whereas the specific activity of VppD is ∼10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation.