Microorganisms such as Pseudomonas putida play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in Pseudomonas putida, we measured differential protein abundance levels with MS-based spectral counting in P. putida S16 grown on nicotine or glycerol, a non-repressive carbon source. In silico analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase (spmA), molybdopterin dehydrogenase (spmB), and a (2Fe-2S)-binding ferredoxin (spmC) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase (nicA2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds.

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 ϳ10-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. N icotine, which easily passes biological membranes, is significantly harmful to humans and is considered a potentially addictive drug (1, 2). Large quantities of tobacco waste with high concentrations of nicotine are generated annually by cigarette and cigar manufacturing (3). Due to its water solubility, nicotine can easily spread in the environment, emerging as a threat to public health (3). As a result, the U.S. Environmental Protection Agency classified nicotine as a "toxic release inventory" chemical in 1994 (4). Microbes play significant roles in removing nicotine from the environment, and in the meantime, they serve as the catalysts for biotransformation. Nicotine could potentially be used for the production of value-added chemicals. 6-Hydroxy-3-succinoylpyridine (HSP), an important precursor for the synthesis of compounds with biological activities, was efficiently produced by an engineered nicotine-degrading strain (5). Therefore, the study of the mechanism of nicotine degradation provides useful information for both bioremediation and biocatalysis.Many microorganisms that use various nicotine degradation pathways have been isolated from the environment, including Arthrobacter (6), Pseudomonas (7, 8), Ochroba...

Nicotine is an important chemical compound in nature that has been regarded as an environmental toxicant causing various preventable diseases. Several bacterial species are adapted to decompose this heterocyclic compound, including Pseudomonas and Arthrobacter. Pseudomonas putida S16 is a bacterium that degrades nicotine through the pyrrolidine pathway, similar to that present in animals. The corresponding late steps of the nicotine degradation pathway in P. putida S16 was first proposed and demonstrated to be from 2,5-dihydroxy-pyridine through the intermediates N-formylmaleamic acid, maleamic acid, maleic acid, and fumaric acid. Genomics of strain S16 revealed that genes located in the largest genome island play a major role in nicotine degradation and may originate from other strains, as suggested by the constructed phylogenetic tree and the results of comparative genomic analysis. The deletion of gene hpo showed that this gene is essential for nicotine degradation. This study defines the mechanism of nicotine degradation.

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.

Background:The biochemical mechanism of nicotine biodegradation is important. Results: An NADH-dependent and FAD-containing hydroxylase (HspB) for nicotine degradation was purified and mechanistically characterized. Conclusion: The hydroxylase is crucial for nicotine degradation by Pseudomonas putida. Significance: The novel HspB provides a sound basis for future studies aimed at a better understanding of the molecular principles of nicotine degradation.