Nicotine Oxidase Research Articles

Improving the kinetic parameters of nicotine oxidizing enzymes by homologous structure comparison and rational design

Demand exists for a nicotine oxidase enzyme with high catalytic efficiency for a variety of applications including the in vivo detection of nicotine, therapeutic enzymatic blockade of nicotine from the CNS, and inactivation of toxic industrial wastes generated in the manufacture of tobacco products. Nicotine oxidase enzymes identified to date suffer from low efficiency, exhibiting either a high kcat or low Km, but not both. Here we present the crystal structure of the (S)-6-hydroxy-nicotine oxidase from Shinella sp HZN7 (NctB), an enzyme that oxidizes (S)-nicotine with a high kcat (>1 s−1), that possesses remarkable structural and sequence similarity to an enzyme with a nanomolar Km for (S)-nicotine, the (S)-nicotine oxidase from Pseudomonas putidia strain S16 (NicA2). Based on a comparison of our NctB structure and the previously published crystal structure of NicA2, we successfully employed a rational design approach to increase the rate of oxidative turnover of the NicA2 enzyme by ∼25% (0.011 s−1 to 0.014 s−1), and reduce the Km of the NctB protein by approximately 34% (940 μM–622 μM). While modest, these results are a step towards engineering a nicotine oxidase with kinetic parameters that fulfill the functional requirements of biosensing, waste remediation, and therapeutic applications.

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.

Impact of Nicotine Metabolism on Nicotine’s Pharmacological Effects and Behavioral Responses: Insights from a Cyp2a(4/5)bgs-Null Mouse

Nicotine metabolism is believed to affect not only nicotine's pharmacological effects but also nicotine addiction. As a key step toward testing this hypothesis, we have studied nicotine metabolism and nicotine's pharmacological and behavioral effects in a novel knockout mouse model [named Cyp2a(4/5)bgs-null] lacking a number of cytochrome P450 genes known to be or possibly involved in nicotine metabolism, including two Cyp2a and all Cyp2b genes. We found that, compared with wild-type mice, the Cyp2a(4/5)bgs-null mice showed >90% decreases in hepatic microsomal nicotine oxidase activity in vitro, and in rates of systemic nicotine clearance in vivo. Further comparisons of nicotine metabolism between Cyp2a(4/5)bgs-null and Cyp2a5-null mice revealed significant roles of both CYP2A5 and CYP2B enzymes in nicotine clearance. Compared with the behavioral responses in wild-type mice, the decreases in nicotine metabolism in the Cyp2a(4/5)bgs-null mice led to prolonged nicotine-induced acute pharmacological effects, in that null mice showed enhanced nicotine hypothermia and antinociception. Furthermore, we found that the Cyp2a(4/5)bgs-null mice developed a preference for nicotine in a conditioned place preference test, a commonly used test of nicotine's rewarding effects, at a nicotine dose that was 4-fold lower than what was required by wild-type mice. Thus, CYP2A/2B-catalyzed nicotine clearance affects nicotine's behavioral response as well as its acute pharmacological effects in mice. This result provides direct experimental support of the findings of pharmacogenetic studies that suggest linkage between rates of nicotine metabolism and smoking behavior in humans.

Cloning of a Novel Nicotine Oxidase Gene from Pseudomonas sp. Strain HZN6 Whose Product Nonenantioselectively Degrades Nicotine to Pseudooxynicotine

Pseudomonas sp. strain HZN6 utilizes nicotine as its sole source of carbon, nitrogen, and energy. However, its catabolic mechanism has not been elucidated. In this study, self-formed adaptor PCR was performed to amplify the upstream sequence of the pseudooxynicotine amine oxidase gene. A 1,437-bp open reading frame (designated nox) was found to encode a nicotine oxidase (NOX) that shows 30% amino acid sequence identity with 6-hydroxy-l-nicotine oxidase from Arthrobacter nicotinovorans. The nox gene was cloned into a broad-host-range cloning vector and transferred into the non-nicotine-degrading bacteria Escherichia coli DH5α (DH-nox) and Pseudomonas putida KT2440 (KT-nox). The transconjugant KT-nox obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine, while DH-nox did not. Reverse transcription-PCR showed that the nox gene is expressed in both DH5α and KT2440, suggesting that additional factors required for nicotine degradation are present in a Pseudomonas strain(s), but not in E. coli. The mutant of strain HZN6 with nox disrupted lost the ability to degrade nicotine, but not pseudooxynicotine. These results suggested that the nox gene is responsible for the first step of nicotine degradation. The (RS)-nicotine degradation results showed that the two enantiomers were degraded at approximately the same rate, indicating that NOX does not show chiral selectivity. Site-directed mutagenesis revealed that both the conserved flavin adenine dinucleotide (FAD)-binding GXGXXG motif and His456 are essential for nicotine degradation activity.

Characterization of a Cyp2a(4/5)bgs‐null Mouse Model: Role of CYP2A and CYP2B in Nicotine Metabolism

Nicotine metabolism is believed to affect not only nicotine's pharmacological effects, but also nicotine addiction. As a key step toward testing this hypothesis, we have produced knockout mouse models targeting P450 genes possibly involved in nicotine metabolism, including Cyp2a and Cyp2b. Our recent study of a Cyp2a5‐null mouse confirmed that, although CYP2A5 plays a major role in systemic clearance of nicotine and cotinine, other P450 enzymes may also have significant roles. The aim of this study is to determine, using a newly generated Cyp2a(4/5)bgs‐null mouse model, whether hepatic P450 enzymes (other than CYP2A5) encoded by the Cyp2a(4/5)bgs gene cluster, including CYP2A4 and the various CYP2B isoforms, contribute to nicotine metabolism in vivo. We found that, compared to the Cyp2a5‐null mice, the Cyp2a(4/5)bgs‐null mice showed ~60% decreases in hepatic microsomal nicotine oxidase activity in vitro, and in rates of systemic nicotine clearance in vivo. The decreases in nicotine metabolism in the Cyp2a(4/5)bgs‐null mice were accompanied by prolonged nicotine‐induced hypothermia and analgesia, compared to the behavioral responses in wild‐type mice. Thus, both CYP2A and CYP2B contribute to nicotine clearance in mice. The Cyp2a(4/5)bgs‐null and Cyp2a5‐null mouse models will be valuable for studies on the impact of genetic variations in nicotine metabolism, as occur in humans, on nicotine dependence.

Role of mouse CYP2A5 in nicotine clearance and testosterone homeostasis

CYP2A5, a mouse P450 enzyme, is expressed abundantly in the olfactory mucosa (OM), and in other tissues, including the liver and the lateral nasal gland (LNG). To determine the biological and pharmacological functions of CYP2A5 in vivo, we have generated a Cyp2a5‐null mouse. Homozygous Cyp2a5‐null mice are viable and fertile. OM microsomes from Cyp2a5‐null mice had significantly lower activities in the metabolism of testosterone (T) than did OM microsomes from wild‐type (WT) mice. In contrast, hepatic microsomes from Cyp2a5‐null mice showed only small decreases in the overall rates of formation of T metabolites, compared to hepatic microsomes from WT mice. Serum T levels were not altered by the loss of CYP2A5 function in male mice, a result confirming that CYP2A5 does not play a major role in systemic T clearance in vivo. Nevertheless, loss of CYP2A5 was accompanied by significant increases in tissue levels of T in the LNG, a gland important for nasal mucus secretion and production of odorant binding proteins. The Cyp2a5‐null mice also showed decreased clearance of nicotine, with significant increases in the half life and AUC values for serum nicotine, compared with WT mice. These findings indicate that CYP2A5 is the major nicotine oxidase in the mouse liver, and that CYP2A5 plays an important role in the regulation of T levels in the LNG of male mice. (Supported in part by NIH grant ES‐07462)

Two closely related pathways of nicotine catabolism in Arthrobacter nicotinovorans and Nocardioides sp. strain JS614

A virtually identical nicotine catabolic pathway including the heterotrimeric molybdenum enzyme nicotine and 6-hydroxy-pseudo-oxynicotine dehydrogenase, 6-hydroxy-L: -nicotine oxidase, 2,6-dihydroxy-pseudo-oxynicotine hydrolase, and 2,6-dihydroxypyridine hydroxylase have been identified in A. nicotinovorans and Nocardioides sp. JS614. Enzymes catalyzing the same reactions and similar protein antigens were detected in the extracts of the two microorganisms. Nicotine blue and methylamine, two end products of nicotine catabolism were detected in the growth medium of both bacterial species. Nicotine catabolic genes are clustered on pAO1 in A. nicotinovorans, but located chromosomally in Nocardioides sp. JS614.

Limitation of cigarette consumption by CYP2A6*4, *7 and *9 polymorphisms

The whole gene deletion CYP2A6*4, the defect of the main nicotine oxidase, contributes to limiting lifelong and daily cigarette consumption. However, the effects on smoking habits of CYP2A6*7 and *9, two major functional polymorphisms common in Asian populations, have not been reported. The present study examined the relationship between polymorphisms *4, *7 and *9 with the smoking habits of 200 Japanese smokers who visited the Keio University Hospital (Tokyo, Japan). The allele frequencies of *1 (wild type), *4, *7 and *9 were 52, 17, 11 and 20%, respectively. When the three polymorphisms were considered simultaneously, the percentages of homozygous wild type, heterozygote, and homozygous mutants and compound heterozygotes were 26.0, 52.5 and 21.5%, respectively. Homozygous mutants and compound heterozygotes (n = 43) smoked fewer cigarettes daily than heterozygotes (n = 105) and homozygous wild-type individuals (n = 52). Smokers with *7/*7, *9/*9 or *7/*9 had lower daily cigarette consumption than smokers with *1/*1. In conclusion, polymorphisms *4, *7 and *9 of CYP2A6 were detected in approximately three out of four Japanese smokers, and their daily cigarette consumption was genetically modulated by these functional polymorphisms.

Identification of a Single Nucleotide Polymorphism in the TATA Box of the CYP2A6 Gene: Impairment of Its Promoter Activity

Human cytochrome P450 2A6 (CYP2A6) constitutes the major nicotine oxidase, and large interindividual differences are seen in the levels of this enzyme, to a great extent caused by the distribution of several different polymorphic gene variants mainly located in the open reading frame (ORF). In the present study, we report a common polymorphism located in the 5′ flanking region of CYP2A6 affecting its expression. DHPLC analysis and complete sequence of the open reading frame of the gene from a Turkish individual revealed a −48T > G substitution disrupting the TATA box. Using dynamic allele-specific hybridization (DASH), genotyping of this novel variant (named CYP2A6*9) was carried out in 116 Swedish, 132 Turkish, and 102 Chinese subjects, and the allele frequencies were found to be 5.2, 7.2, and 15.7%, respectively. The significance of the polymorphism was investigated by the construction of luciferase reporter plasmids containing 135 or 500 bp of the 5′-upstream region of the gene transfected into human hepatoma B16A2 cells. The constructs carrying the −48T > G mutation were only expressed at about 50% of the wild-type alleles. It is concluded that the CYP2A6*9 allele might be one of the most common CYP2A6 variants in Caucasians that alters the levels of enzyme expression.

39] Determining covalent flavinylation

Publisher Summary Covalent flavoproteins are found in bacteria, plants, and mammals. Many of them are engaged in the dissimilation of organic material. In mammalian species, the number of covalent flavoproteins appears to be limited. Four covalent flavoproteins have been distinguished in rat liver mitochondria and identified as succinate dehydrogenase, mono-amine oxidase, sarcosine dehydrogenase, and dimethylglycine dehydrogenase; further mammalian covalent flavoproteins are L -gulono- γ -lactone oxidase in rat liver microsomes and pipecolic acid oxidase in monkey liver peroxisomes. Flavinylation may occur co- or posttranslationally, involving the mature apoenzyme or a precursor protein. The mechanism of covalent flavin–protein attachment has first been elucidated for the histidyl(N-3)–8 α -flavin adenine dinucleotide (FAD) linkage in 6-hydroxy- D -nicotine oxidase (6HDNO). The most abundant type of covalent flavin bond involves a histidine residue of the polypeptide chain. Histidine may be bound at its N-1 or N-3 position to the 8 α -methyl group of the isoalloxazine ring forming secondary amines. The 8 α -position of the flavin was also found attached to the phenolic OH group of a tyrosyl residue in p -cresol hydroxymethylase (4-cresol dehydrogenase). The flavoproteins with flavin bound to a cysteinyl residue include two different binding types: polypeptides bound by thioether linkage to the 8-methyl group or to carbon atom 6 of the isoalloxazine ring. They differ in chemical and physical properties from the His-and Tyr-attached riboflavin species.

Effects of aging on acute toxicity of nicotine in rats.

Acute toxicity of nicotine was examined in old (24 months) and young (6 weeks) Wistar rats. There were no significant age differences in the mortality and convulsive responses induced by an intraperitoneal injection of nicotine (24.5 mg/kg). The lethal nicotine levels in blood and cortex and the latent period to death in old rats were larger than those in young rats. Cortical and blood nicotine levels 15 min. after the nicotine injection in old rats were also higher than those in young rats. Nicotine significantly increased dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid levels in striatum and hippocampus in young rats, but not those in old rats. Moreover, nicotine-induced elevations in blood levels of corticosterone in old rats were also less than those in young rats. On the other hand, the cytochrome P-450 content, the nicotine oxidase activity and the flavin-containing monooxygenase activity in liver showed age-related decreases in the young, the middle-aged (12 months) and the old rats. These results indicate that the acute toxicity of nicotine in old rats reflects the decreases in hepatic nicotine metabolism and in brain sensitivity to nicotine.

Rabbit nasal cytochrome P-450 NMa has high activity as a nicotine oxidase

Rabbit nasal olfactory and respiratory microsomes demonstrate high activity toward [ 3H]-(S)-nicotine, with specific activities of 22.2 and 6.5 nmol/min/mg protein, respectively. The major metabolite produced is (S)-nicotine Δ I′,5′-iminium ion, with lesser amounts of nornicotine and the N′-oxide. Reconstitution of the rabbit nasal microsomal system with cytochromes P-450 NMa and NMb indicated that only P-450 NMa has significant activity toward nicotine, and the metabolite profile and turnover are similar to that observed with nasal microsomes. The low K m (35 μM) and high V max (28 min −1) suggest that a significant portion of inhaled nicotine is metabolized by nasal tissues in the rabbit.

Binding of FAD to 6-hydroxy-D-nicotine oxidase apoenzyme prevents degradation of the holoenzyme.

Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed E. coli cells grown at 30 degrees C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37 degrees C. This effect did not depend on the promoter used for expression of the cloned gene in E. coli, nor was it an effect of 6-HDNO mRNA instability at 37 degrees C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo-6-HDNO to proteolytic attack at 37 degrees C was responsible for the observed phenomenon. Extracts from cells grown at 37 degrees C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30 degrees C. The 6-HDNO polypeptide is covalently modified by attachment of the cofactor FAD to a histidine residue. It could be shown that covalent flavinylation of the apoenzyme in vitro, i.e. formation of holoenzyme, by incubation of cell extracts with FAD and phosphoenolpyruvate protected the 6-HDNO polypeptide from degradation at 37 degrees C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.

Cytochrome P-450-dependent nicotine oxidation by liver microsomes of guinea pigs: Immunochemical evidence with antibody against phenobarbital-inducible cytochrome P-450

When guinea pigs were treated with phenobarbital (PB), the specific activity of liver microsomal nicotine oxidase increased by 42%. PB-inducible cytochrome P-450 (PB-P-450) was purified to homogeneity from liver microsomes of PB-treated guinea pigs. Purified PB-P-450 catalyzed nicotine oxidation when reconstituted with NADPH-P-450 reductase and phospholipid system. Antibody prepared against the purified PB-P-450 formed single precipitation lines with both purified PB-P-450 and microsomal components in livers of PB-treated guinea pigs, and both precipitation lines fused. The antibody against PB-P-450 strongly inhibited nicotine oxidation in the reconstituted system. The antibody also inhibited liver microsomal nicotine oxidase activities in PB-treated and untreated guinea pigs by about 30% and less than 5% respectively. About 45% of total P-450 in liver microsomes of PB-treated guinea pigs was precipitated by the antibody. These results show that PB-P-450 participates in liver microsomal nicotine oxidation in PB-treated guinea pigs but not in untreated control animals.

ラットのサーカディアンリズムにおよぼすニコチン自由摂取の影響

Male Wistar rats, aged 4 weeks, were kept in a temperature controlled room with a 12 hr light-dark cycle and given food and water ad libitum. The nicotine-treated group of rats was given water containing nicotine, which was estimated to be 10 mg/kg/day, for 40 days. Spontaneous motor activities, drinking activities and serum corticosterone levels showed circadian rhythms characteristic of a nocturnal animal in both the control and the nicotine-treated groups. As compared to the control group, however, the nicotine-treated group showed an increase in ambulatory activities, a decrease in drinking activities and a diminution of weight gain. In comparison with diurnal variations, serum corticosterone levels and liver nicotine oxidase activities increased in the nicotine-treated group during the dark period. However, the pattern of circadian rhythms characteristic of a nocturnal animal were not altered.

P-321) - Nicotine oxidase in liver microsomes of guinea pigs

P-321) - Nicotine oxidase in liver microsomes of guinea pigs

Quantitation of immunoadsorbed flavoprotein oxidases by luminol-mediated chemiluminescence.

The detection of the flavoenzymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase at the sub-femtomol level was achieved by coupling the reaction of the immunoadsorbed proteins to the peroxidase-catalysed oxidation of luminol. The H2O2-producing oxidases retained their full activity when bound to the respective immobilized antibodies. This fact allowed the concentration of the enzymes from very dilute solutions and the quantitative assay of their activities in the microU range. Due to strict stereoselectivity and the absence of immunological cross-reactivity, the two flavoproteins could be determined in the same solution. This method was used to measure the 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase activities in Escherichia coli RR1 and different Arthrobacter strains cultured under non-inducing conditions. The same activity ratio of 6-hydroxy-L-nicotine oxidase/6-hydroxy-D-nicotine oxidase as in D L-nicotine-induced cells of A. oxidans was observed in non-induced wild type and in riboflavin-requiring (rf-) mutant cells of this aerob.

Nicotine metabolism and biorhythms

Previously we have reported that among various animals injected i.p. with nicotine in the daytime, the induction of the drug metabolizing enzyme activities were observed in the liver of dogs, but not in the liver of rats; and when nicotine with drinking water was given to rats ad libitum, the induction of these enzyme activities were observed. Then we made some investigations into biorhythms using male Wistar rats under a 14 : 10 light-dark cycle. The dark phases began at 8:00 p.m. and ended at 6:00 a.m.. Illumination (8:00 a.m. to 6:00 p.m.) was provided by fluorescent light (approximate 100 Lux), and the light was changed gradually for dawn and dusk. Sampling was performed every 4 hr over a 24 hr period. Circadian changes were demonstrated concerning the levels of plasma corticosterone and brain 5-hydroxytryptamine and S-hydroxyindole acetate. Drinking water behavior activities and spontaneous motor activities of rats synchronized with the light-dark alternation, thus showing regular 24-hr rhythms. It was also observed that there were circadian rhythms of the drug metabolizing enzyme activities of the liver, including nicotine oxidase and benzopyrene- and acetylaminofluorene-hydroxylases; these revealed higher activities in the dark than in the light. On the other hand, acute mortality to i.p. injection of nicotine showed rhythmic changes; these were higher in the dark than in the light. These results suggest that it is important to take great care in choosing the time at which to take sample materials and to administer drugs when nocturnal animals such as rats are emploied.

Difference of nicotine metabolizing enzyme activities in various species (17).

Nicotine is known to be metabolized in liver of animals. In this report, induction of nicotine oxidase activity and nicotine binding to purified phenobarbital(PB)-inducible cytochrome P-450 were investigated. Pretreatment of guinea pigs with PB increased both specific content of cytochrome P-450 and specific activity of nicotine oxidase, but the activity of the enzyme per cytochrome P-450 remained unchanged. PB-in-ducible cytochrome P-450 was purified from PB-pretreated guinea pigs by chromatography on octylamino-Sepharose 4B, DE-52 and hydroxyapatite column. The purified enzyme showed a single protein-staining band on SDS-polyacrylamide gel with apparent molecular weight of 51,000 and 19.5 nmole/mg protein of specific content. Addition of nicotine to purified PB-inducible cytochrome P-450 caused a shift of maximum of Soret peak toward the red approximately 2 nm. The difference spectrum produced by nicotine showed a type 2 spectral change with a peak at 429 nm and a trough at 4 08 nm. Lineweaver-Burk plot of the spectral change showed two slopes. The spectral dissociation constants were 0.44 and 5.0 mM. These results suggest that PB-inducible cytochrome P-450 oxidizes nicotine in guinea pigs.

Heterogeneity of hepatic nicotine oxidase

When rats were pretreated with 3-methylcholanthrene or β-naphthoflavone, hepatic nicotine oxidase activity per cytochrome P-448 molecule decreased, but the specific activity of the enzyme remained unchanged. After phenobarbital pretreatment, the specific activity of nicotine oxidase increased while the activity of the enzyme per cytochrome P-450 molecule decreased. α-Naphthoflavone selectively inhibited the activities of phenobarbital-induced nicotine oxidase and constitutive form(s) of the enzyme. These results show that phenobarbital-induced cytochrome P-450 and constitutive forms(s) of the enzyme may be active in hepatic nicotine oxidation.