Structural Analysis Provides Mechanistic Insight into Nicotine Oxidoreductase from <i>Pseudomonas putida</i>

Tararina, Margarita; Janda, Kim D.; Allen, Karen N.

doi:10.1021/acs.biochem.6b00963

Cited by 20 publications

(38 citation statements)

References 30 publications

(79 reference statements)

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“…2c (Table 2) suggest that at least for one variant (A107R) enhanced k cat comes at the expense of increased K m (the enzyme concentration needed to attain 50% of the maximum rate of catalysis, V max ). As a validation of the kinetic modeling of low nicotine progress curves, we found wt NicA2 had an apparent k cat / K m of 6.4 × 10 4 M − 1 s − 1 in close agreement with prior reports [26]. The variants shown in Fig.…”

Section: Resultssupporting

confidence: 90%

“…In an effort to eliminate any non-essential bacterial sequence (including a specific in silico predicted T-cell epitope sequence [29]) to reduce immunogenic risk, a deletion construct was made removing the first 50 N-terminal residues which appeared unstructured in the crystal structure [26], resulting in the construct NicA2Δ50. Figure 1b shows the Amplex Red assay results (shown as calculated slopes of Relative Fluorescence Units (RFU) as a function of time) for purified NicA2 and NicA2Δ50 enzymes.…”

Section: Resultsmentioning

confidence: 99%

“…While the structure of NicA2 has been elucidated [26, 27], very limited knowledge about the role and importance of individual residues in catalytic activity exists, and to date only 2 rationally designed mutations, T381V and T250V/T381V, have been reported in the literature; each showing a decrease in catalytic efficiency ( k cat / K m ) in vitro [27]. We have taken the approach of screening libraries randomizing all active site positions to start collecting empirical data on the effect on enzymatic activity by mutating various residues.…”

Section: Introductionmentioning

confidence: 99%

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Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction

Thisted¹,

Biesová²,

Walmacq³

et al. 2019

BMC Biotechnol

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Background:Smoking and tobacco use continue to be the largest preventable causes of death globally. A novel therapeutic approach has recently been proposed: administration of an enzyme that degrades nicotine, the main addictive component of tobacco, minimizing brain exposure and reducing its reinforcing effects. Pre-clinical proof of concept has been previously established through dosing the amine oxidase NicA2 from Pseudomonas putida in rat nicotine self-administration models of addiction. Results: This paper describes efforts towards optimizing NicA2 for potential therapeutic use: enhancing potency, improving its pharmacokinetic profile, and attenuating immunogenicity. Libraries randomizing residues located in all 22 active site positions of NicA2 were screened. 58 single mutations with 2-to 19-fold enhanced catalytic activity compared to wt at 10 μM nicotine were identified. A novel nicotine biosensor assay allowed efficient screening of the many primary hits for activity at nicotine concentrations typically found in smokers. 10 mutants with improved activity in rat serum at or below 250 nM were identified. These catalytic improvements translated to increased potency in vivo in the form of further lowering of nicotine blood levels and nicotine accumulation in the brains of Sprague-Dawley rats. Examination of the X-ray crystal structure suggests that these mutants may accelerate the rate limiting re-oxidation of the flavin adenine dinucleotide cofactor by enhancing molecular oxygen's access. PEGylation of NicA2 led to prolonged serum half-life and lowered immunogenicity observed in a human HLA DR4 transgenic mouse model, without impacting nicotine degrading activity.Conclusions: Systematic mutational analysis of the active site of the nicotine-degrading enzyme NicA2 has yielded 10 variants that increase the catalytic activity and its effects on nicotine distribution in vivo at nicotine plasma concentrations found in smokers. In addition, PEGylation substantially increases circulating half-life and reduces the enzyme's immunogenic potential. Taken together, these results provide a viable path towards generation of a drug candidate suitable for human therapeutic use in treating nicotine addiction.

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction

Thisted¹,

Biesová²,

Walmacq³

et al. 2019

BMC Biotechnol

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“…Preliminary studies of NicA2 have characterized the in vitro properties of this enzyme pertinent to its potential therapeutic use [ 8 ]. NicA2 is a 52.5 kDa protein which, when expressed in E. coli , is complexed with flavin adenine dinucleotide (FAD, a redox co-factor) as indicated by the recently published high-resolution crystal structure [ 18 ], and remains catalytically active after isolation without addition of any other components. NicA2 has high catalytic activity with k cat of 0.013 s − 1 , K m of 0.092 μM, and k cat / K m = 1.4 × 10 5 s − 1 • M − 1 (37 °C), and it rapidly degrades nicotine in vitro at nicotine concentrations representative of serum concentrations in heavy smokers [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

The nicotine-degrading enzyme NicA2 reduces nicotine levels in blood, nicotine distribution to brain, and nicotine discrimination and reinforcement in rats

et al. 2018

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Background: The bacterial nicotine-degrading enzyme NicA2 isolated from P. putida was studied to assess its potential use in the treatment of tobacco dependence. Results: Rats were pretreated with varying i.v. doses of NicA2, followed by i.v. administration of nicotine at 0.03 mg/kg. NicA2 had a rapid onset of action reducing blood and brain nicotine concentrations in a dose-related manner, with a rapid onset of action. A 5 mg/kg NicA2 dose reduced the nicotine concentration in blood by > 90% at 1 min after the nicotine dose, compared to controls. Brain nicotine concentrations were reduced by 55% at 1 min and 92% at 5 min post nicotine dose. To evaluate enzyme effects at a nicotine dosing rate equivalent to heavy smoking, rats pretreated with NicA2 at 10 mg/kg were administered 5 doses of nicotine 0.03 mg/kg i.v. over 40 min. Nicotine levels in blood were below the assay detection limit 3 min after either the first or fifth nicotine dose, and nicotine levels in brain were reduced by 82 and 84%, respectively, compared to controls. A 20 mg/kg NicA2 dose attenuated nicotine discrimination and produced extinction of nicotine self-administration (NSA) in most rats, or a compensatory increase in other rats, when administered prior to each daily NSA session. In rats showing compensation, increasing the NicA2 dose to 70 mg/kg resulted in extinction of NSA. An enzyme construct with a longer duration of action, via fusion with an albumin-binding domain, similarly reduced NSA in a 23 h nicotine access model at a dose of 70 mg/kg. Conclusions: These data extend knowledge of NicA2's effects on nicotine distribution to brain and its ability to attenuate addiction-relevant behaviors in rats and support its further investigation as a treatment for tobacco use disorder.

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“…Previous enzymatic studies on this family have generally favored a reaction mechanism involving direct hydride transfer. Recently, NicA2 from P. putida S16 has even been proposed as a resource for the development of novel nicotine addiction therapies ( 29 , 30 ); the X-ray crystal structures of NicA2 alone and NicA2 in complex with nicotine have been reported at resolutions of 2.51 Å and 2.65 Å, respectively ( 31 , 32 ).…”

Section: Introductionmentioning

confidence: 99%

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

Tang

Zhang

et al. 2020

mBio

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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.

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Structural Analysis Provides Mechanistic Insight into Nicotine Oxidoreductase from Pseudomonas putida

Cited by 20 publications

References 30 publications

Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction

Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction

The nicotine-degrading enzyme NicA2 reduces nicotine levels in blood, nicotine distribution to brain, and nicotine discrimination and reinforcement in rats

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

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