Biology subjects

Stull, F.

Publications and source records attributed to Stull, F..

5 recordsLinked to original sources

bioRxiv2026

Isolation of oxygen-dependent nicotine- and pseudooxynicotine-metabolizing enzymes

NicA2 is a flavin-bound amine dehydrogenase from Pseudomonas putida S16 that converts nicotine to the pharmacologically inactive N-methylmyosmine. In animal models of nicotine addiction, injection of NicA2 can decrease nicotine-seeking behavior 10-fold. Accordingly, NicA2-related enzymes have been investigated as smoking-cessation therapeutics. However, efficient catalysis by NicA2 in Pseudomonas putida relies on electron transfer to CycN, a cytochrome c, and not directly to O2. Impractically high amounts of NicA2 are thus necessary to achieve a pharmacological effect in the absence of CycN. Directed evolution has improved the ambient-O2 value of kcat from 0.007 s-1 to 1 s-1 for NicA2, but further improvements have been challenging. Here, we identify a strain of Peribacillus frigoritolerans NIC8 which encodes two flavin amine oxidoreductases, Ncox and Pnox. In the presence of oxygen, Ncox and Pnox act on nicotine and pseudooxynicotine respectively with apparent kcat values of 7.7 s-1 and 3.9 s-1. Transient kinetics establishes bimolecular rate constants of 51100 M-1s-1 and 81000 M-1s-1 for the half-reactions between Ncox and O2 and between Pnox and O2 respectively, consistent with Ncox and Pnox being bona-fide oxidases. Transcriptomics shows enhanced expression of Ncox and Pnox under nicotine-dependent growth as well as supporting the identification of downstream enzymes. Phylogenetic analysis suggests that Ncox and Pnox arose out of repurposing of homologous enzymes found in Bacillus species. The enzymes we describe may be useful for the development of nicotine addiction therapeutics and for bioconversion of nicotine in waste streams.

biochemistry↗

bioRxiv2025

Role of glutamate 292 and lysine 331 in catalysis for the flavoenzyme (S)-6-hydroxynicotine oxidase from Shinella sp. HZN7

The flavoenzyme NctB from Shinella sp. HZN7 catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine concomitant with dioxygen reduction, which is the same chemistry catalyzed by the well-studied flavoenzyme L-6-hydroxynicotine oxidase (LHNO) from Paenarthrobacter nicotinovorans. However, while both enzymes are members of the flavoprotein amine oxidoreductase (FAO) family, they share only 26% sequence identity and are evolutionarily distant. Furthermore, nearly all FAOs (including LHNO) have a conserved lysine proximal to N5 of the flavin that is known to promote the reaction with O2 in the oxidative half-reaction, yet NctB, unusually, has a glutamate (Glu292) at this position. We report here using transient kinetics that NctB reacts rapidly with dioxygen in the oxidative half-reaction despite lacking the conserved lysine associated with promoting the reaction with O2 in FAO family enzymes. Mutagenesis reveals that a lysine derived from a different sequence position (Lys331) likely accelerates the reaction with dioxygen in NctB, as the K331M mutation results in a 1400-fold decrease in rate constant for reaction with O2. Glu292 forms a salt bridge with Lys331 in the structure of NctB, and a E292T mutation results in a [~]80-fold decrease in rate constant for reaction with O2, suggesting that Glu292 optimizes the positioning and/or properties of Lys331 to promote dioxygen activation. Analysis of pH-rate effects in NctB shows similar pH profiles as in LHNO despite having differences in active site structure. These results indicate that NctB and LHNO convergently evolved to have the same enzymatic function.

biochemistry↗

bioRxiv2025

Ancestral evolution of oxidase activity in a class of (S)-nicotine and (S)-6-hydroxynicotine degrading flavoenzymes

Reduced flavin cofactors have the innate ability to reduce molecular oxygen to hydrogen peroxide. Flavoprotein oxidases turbocharge the reaction of their flavin cofactor with oxygen whereas flavoprotein dehydrogenases generally suppress it, yet our understanding of how these two enzyme classes control this reactivity remains incomplete. Here we used ancestral sequence reconstruction and biochemical characterization to retrace the evolution of oxidase activity in a lineage of nicotine/6-hydroxynicotine degrading enzymes of the flavoprotein amine oxidase superfamily. Our data suggest that the most ancient ancestor that gave rise to this lineage was a dehydrogenase, and that oxidase activity emerged later from within this group of dehydrogenases. We have identified the key amino acid replacements responsible for this emergence of oxidase activity, which, remarkably, span the entire protein structure. Molecular dynamics simulations indicate that this constellation of substitutions decreases the global dynamics of the protein in the evolution of oxidase function. This coincides with a dramatic restriction in the movement of a lysine residue in the active site, which more optimally positions it in front of the flavin to promote the reaction with O2. Our results demonstrate that sites distant from the flavin microenvironment can help control flavin-oxygen reactivity in flavoenzymes by modulating the conformational space and dynamics of the protein and catalytic residues in the active site.

biochemistry↗

bioRxiv2024

G-quadruplexes catalyze protein folding by reshaping the energetic landscape

Many proteins have slow folding times in vitro that are physiologically untenable. To combat this challenge, ATP-dependent chaperonins are thought to possess the unique ability to catalyze protein folding. Performing quantitative model selection using protein folding and unfolding data, we here show that short nucleic acids containing G-quadruplex (G4) structure can also catalyze protein folding. Performing the experiments as a function of temperature demonstrates that the G4 reshapes the underlying driving forces of protein folding. As short nucleic acids can catalyze protein folding without the input of ATP, the ability of the cell to fold proteins is far higher than previously anticipated. Significance StatementHow folding of proteins occurs en masse in the cell is still a daunting unsolved problem. Many proteins have complicated and difficult folding trajectories, with in vitro folding times that are physiologically untenable. The acceleration of protein folding to physiologically relevant timescales is a biologically essential function thought to be accomplished by a small set of ATP-dependent chaperonins. In this work, we surprisingly show that small nucleic acid sequences containing G-quadruplexes can catalyze protein folding and reshape protein folding energy landscapes. As a result, the capacity for accelerating protein folding in the cells is far higher than previously suggested, potentially explaining the accommodation of large number of proteins with physiologically unreasonable folding times.

biophysics↗

bioRxiv2024

Rational Design of a Flavoenzyme for Aerobic Nicotine Catabolism

Enzymatic therapy with nicotine-degrading enzyme is a new strategy in treating nicotine addiction, which can reduce nicotine concentrations and weaken withdrawal in the rat model. However, when O2 is used as the electron acceptor, no satisfactory performance has been achieved with one of the most commonly studied and efficient nicotine-catabolizing enzymes, NicA2. To obtain more efficient nicotine-degrading enzyme, we rationally designed and engineered a flavoenzyme Pnao, which shares high structural similarity with NicA2 (RMSD = 1.143 [A]) and efficiently catalyze pseudooxynicotine into 3-succinoyl-semialdehyde pyridine using O2. Through amino acid alterations with NicA2, five Pnao mutants were generated, which can degrade nicotine in Tris-HCl buffer and retained catabolic activity on its natural substrate. Nicotine-1-N-oxide was identified as one of the reaction products. Four of the derivative mutants showed activity in rat serum and Trp220 and Asn224 were found critical for enzyme specificity. Our findings offer a novel avenue for research into aerobic nicotine catabolism and provides a promising method of generating additional nicotine-catalytic enzymes.

molecular biology↗