Flavin Reduction Research Articles

The rapid-reaction kinetics of an electron-bifurcating flavoprotein, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase EtfAB:bcd

We have investigated the kinetic behavior of the electron-bifurcating crotonyl-CoA-dependent NADH: ferredoxin oxidoreductase EtfAB:bcd from Megasphaera elsdenii. The overall behavior of the complex in both the reductive and oxidative half-reactions is consistent with that previously determined for the individual EtfAB and bcd components. This includes an uncrossing of the half-potentials of the bifurcating flavin of the EtfAB component in the course of ferredoxin-reducing catalysis, ionization of the bcd flavin semiquinone and the appearance of a charge transfer complex upon binding of the high potential acceptor crotonyl-CoA. The observed rapid-reaction rates of ferredoxin reduction are independent of [NADH], [crotonyl-CoA] and [ferredoxin], with an observed rate of ∼0.2 s-1, consistent with the observed steady-state kinetics. In enzyme-monitored turnover experiments, an approach to steady-state where the complex’s flavins become reduced but no ferredoxin is generated is followed by a steady-state phase characterized by extensive ferredoxin reduction but little change in overall levels of flavin reduction. The approach to steady-state phase can be eliminated by prior reduction of the complex, in which case there is no lag in the onset of ferredoxin reduction; this is consistent with the et FAD needing to be reduced to the level of the (anionic) semiquinone for bifurcation and concomitant ferredoxin reduction to occur. Single-turnover experiments support this conclusion, with accumulation of the anionic semiquinone of the et FAD apparently required to prime the system for subsequent bifurcation and ferredoxin reduction.

Evolution, structure, and drug-metabolizing activity of mammalian prenylcysteine oxidases

Prenylcysteine oxidases (PCYOXs) metabolize prenylated cysteines produced by protein degradation. They utilize oxygen as a co-substrate to produce free cysteine, an aldehyde, and hydrogen peroxide through the unusual oxidation of a thioether bond. In this study, we explore the evolution, structure, and mechanism of the two mammalian PCYOXs. A gene duplication event in jawed vertebrates originated in these two paralogs. Both enzymes are active on farnesyl- and geranylgeranylcysteine, but inactive on molecules with shorter prenyl groups. Kinetics experiments outline a mechanism where flavin reduction and re-oxidation occur rapidly without any detectable intermediates, with the overall reaction rate limited by product release. The experimentally determined three-dimensional structure of PCYOX1 reveals long and wide tunnels leading from the surface to the flavin. They allow the isoprene substrate to curl up within the protein and position its reactive cysteine group close to the flavin. A hydrophobic patch on the surface mediates membrane association, enabling direct substrate and product exchange with the lipid bilayer. Leveraging established knowledge of flavoenzyme inhibition, we designed sub-micromolar PCYOX inhibitors. Additionally, we discovered that PCYOXs bind and slowly degrade salisirab, an anti-RAS compound. This activity suggests potential and previously unknown roles of PCYOXs in drug metabolism.

Functional and structural characterization of a thermostable flavin reductase from Geobacillus mahadii Geo-05

Flavin reductases play a vital role in catalyzing the reduction of flavin through NADH or NADPH oxidation. The gene encoding flavin reductase from the thermophilic bacterium Geobacillus mahadii Geo-05 (GMHpaC) was cloned, overexpressed in Escherichia coli BL21 (DE3) pLysS, and purified to homogeneity. The purified recombinant GMHpaC (Class II) contains chromogenic cofactors, evidenced by maximal absorbance peaks at 370 nm and 460 nm. GMHpaC stands out as the most thermostable and pH-tolerant flavin reductase reported to date, retaining up to 95 % catalytic activity after incubation at 70 °C for 30 min and maintaining over 80 % activity within a pH range of 2–12 for 30 min. Furthermore, GMHpaC's catalytic activity increases by 52 % with FMN as a co-factor compared to FAD and riboflavin. GMHpaC, coupled with 4-hydroxyphenylacetate-3-monooxygenase (GMHpaB) from G. mahadii Geo-05, enhances the hydroxylation of 4-hydroxyphenylacetate (HPA) by 85 %. The modeled structure of GMHpaC reveals relatively conserved flavin and NADH binding sites. Modeling and docking studies shed light on structural features and amino acid substitutions that determine GMHpaC's co-factor specificity. The remarkable thermostability, high catalytic activity, and general stability exhibited by GMHpaC position it as a promising enzyme candidate for various industrial applications.

Kinetic Characterization and Identification of Key Active Site Residues of the L-Aspartate N-Hydroxylase, CreE.

CreE is a flavin-dependent monooxygenase (FMO) that catalyzes three sequential nitrogen oxidation reactions of L-aspartate to produce nitrosuccinate, contributing to the biosynthesis of the antimicrobial and antiproliferative nautral product, cremeomycin. This compound contains a highly reactive diazo functional group for which the reaction of CreE is essential to its formation. Nitro and diazo functional groups can serve as potent electrophiles, important in some challenging nucleophilic addition reactions. Formation of these reactive groups positions CreE as a promising candidate for biomedical and synthetic applications. Here, we present the catalytic mechanism of CreE and the identification of active site residues critical to binding L-aspartate, aiding in future enzyme engineering efforts. Steady-state analysis demonstrated that CreE is very specific for NADPH over NADH and performs a highly coupled reaction with L-aspartate. Analysis of the rapid-reaction kinetics showed that flavin reduction is very fast, along with the formation of the oxygenating species, the C4a-hydroperoxyflavin. The slowest step observed was the dehydration of the flavin. Structural analysis and site-directed mutagenesis implicated T65, R291, and R440 in the binding L-aspartate. The data presented describes the catalytic mechanism and the active site architecture of this unique FMO.

Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase.

OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.

'Seeing' the electromagnetic spectrum: spotlight on the cryptochrome photocycle.

Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine.

Activation of Coq6p, a FAD Monooxygenase Involved in Coenzyme Q Biosynthesis, by Adrenodoxin Reductase/Ferredoxin.

Adrenodoxin reductase (AdxR) plays a pivotal role in electron transfer, shuttling electrons between NADPH and iron/sulfur adrenodoxin proteins in mitochondria. This electron transport system is essential for P450 enzymes involved in various endogenous biomolecules biosynthesis. Here, we present an in-depth examination of the kinetics governing the reduction of human AdxR by NADH or NADPH. Our results highlight the efficiency of human AdxR when utilizing NADPH as a flavin reducing agent. Nevertheless, akin to related flavoenzymes such as cytochrome P450 reductase, we observe that low NADPH concentrations hinder flavin reduction due to intricate equilibrium reactions between the enzyme and its substrate/product. Remarkably, the presence of MgCl2 suppresses this complex kinetic behavior by decreasing NADPH binding to oxidized AdxR, effectively transforming AdxR into a classical Michaelis-Menten enzyme. We propose that the addition of MgCl2 may be adapted for studying the reductive half-reactions of other flavoenzymes with NADPH. Furthermore, in vitro experiments provide evidence that the reduction of the yeast flavin monooxygenase Coq6p relies on an electron transfer chain comprising NADPH-AdxR-Yah1p-Coq6p, where Yah1p shuttles electrons between AdxR and Coq6p. This discovery explains the previous in vivo observation that Yah1p and the AdxR homolog, Arh1p, are required for the biosynthesis of coenzyme Q in yeast.

Oligomeric Changes Regulate Flavin Transfer in Two-Component FMN Reductases Involved in Sulfur Metabolism.

The FMN reductases (SsuE and MsuE of the alkanesulfonate monooxygenase systems) supply reduced flavin to their partner monooxygenases for the desulfonation of alkanesulfonates. Flavin reductases that comprise two-component systems must be able to regulate both flavin reduction and transfer. One mechanism to control these distinct processes is through changes in the oligomeric state of the enzymes. Despite their similar overall structures, SsuE and MsuE showed clear differences in their oligomeric states in the presence of substrates. The oligomeric state of SsuE was converted from a tetramer to a dimer/tetramer equilibrium in the presence of FMN or NADPH in analytical ultracentrifugation studies. Conversely, MsuE shifted from a dimer to a single tetrameric state with FMN, and the NADPH substrate did not induce a similar oligomeric shift. There was a fast tetramer to dimer equilibrium shift occurring at the dimer/dimer interface in H/D-X investigations with apo SsuE. Formation of the SsuE/FMN complex slowed the tetramer/dimer conversion, leading to a slower exchange along the dimer/dimer interface. The oligomeric shift of the MsuE/FMN complex from a dimer to a distinct tetramer showed a decrease in H/D-X in the region around the π-helices at the dimer/dimer interface. Both SsuE and MsuE showed a comparable and significant increase in the melting temperature with the addition of FMN, indicating the conformers formed by each FMN-bound enzyme had increased stability. A mechanism that supports the different structural shifts is rationalized by the different roles these enzymes play in providing reduced flavin to single or multiple monooxygenase enzymes.

Reaction mechanism and kinetics of the two-component flavoprotein dimethyl sulfone monooxygenase system: using hydrogen peroxide for monooxygenation and substrate cleavage.

The dimethyl sulfone monooxygenase system is a two-component flavoprotein, catalyzing the monooxygenation of dimethyl sulfone (DMSO2 ) by oxidative cleavage producing methanesulfinate and formaldehyde. The reductase component (DMSR) is a flavoprotein with FMN as a cofactor, catalyzing flavin reduction using NADH. The monooxygenase (DMSMO) uses reduced flavin from the reductase and oxygen for substrate monooxygenation. DMSMO can bind to FMN and FMNH- with a Kd of 17.4 ± 0.9 μM and 4.08 ± 0.8 μM, respectively. The binding of FMN to DMSMO is required prior to binding DMSO2 . This also applies to the fast binding of reduced FMN to DMSMO followed by DMSO2 . Substituting reduced DMSR with FMNH- demonstrated the same oxidation kinetics, indicating that FMNH- from DMSR was transferred to DMSMO. The oxidation of FMNH- :DMSMO, with and without DMSO2 did not generate any flavin adducts for monooxygenation. Therefore, H2 O2 is likely to be the reactive agent to attack the substrate. The H2 O2 assay results demonstrated production of H2 O2 from the oxidation of FMNH- :DMSMO, whereas H2 O2 was not detected in the presence of DMSO2 , confirming H2 O2 utilization. The rate constant for methanesulfinate formation determined from rapid-quenched flow and the rate constant for flavin oxidation were similar, indicating that H2 O2 rapidly reacts with DMSO2 , with flavin oxidation as the rate-limiting step. This is the first report of the kinetic mechanisms of both components using rapid kinetics and of a method for methanesulfinate detection using LC-MS.

Dissecting Reaction Mechanisms and Catalytic Contributions in Flavoprotein Fumarate Reductases.

The interconversion between fumarate and succinate is fundamental to the energy metabolism of nearly all organisms. This redox reaction is catalyzed by a large family of enzymes, fumarate reductases and succinate dehydrogenases, using hydride and proton transfers from a flavin cofactor and a conserved Arg side-chain. These flavoenzymes also have substantial biomedical and biotechnological importance. Therefore, a detailed understanding of their catalytic mechanisms is valuable. Here, calibrated electronic structure calculations in a cluster model of the active site of the Fcc3 fumarate reductase were employed to investigate various reaction pathways and possible intermediates in the enzymatic environment and to dissect interactions that contribute to catalysis of fumarate reduction. Carbanion, covalent adduct, carbocation, and radical intermediates were examined. Significantly lower barriers were obtained for mechanisms via carbanion intermediates, with similar activation energies for hydride and proton transfers. Interestingly, the carbanion bound to the active site is best described as an enolate. Hydride transfer is stabilized by a preorganized charge dipole in the active site and by the restriction of the C1-C2 bond in a twisted conformation of the otherwise planar fumarate dianion. But, protonation of a fumarate carboxylate and quantum tunneling effects are not critical for catalysis of the hydride transfer. Calculations also suggest that the driving force for enzyme turnover is provided by regeneration of the catalytic Arg, either coupled with flavin reduction and decomposition of a proposed transient state or directly from the solvent. The detailed mechanistic description of enzymatic reduction of fumarate provided here clarifies previous contradictory views and provides new insights into catalysis by essential flavoenzyme reductases and dehydrogenases.

Mechanism of the Multistep Catalytic Cycle of 6-Hydroxynicotinate 3-Monooxygenase Revealed by Global Kinetic Analysis

The class A flavoenzyme 6-hydroxynicotinate 3-monooxygenase (NicC) catalyzes a rare decarboxylative hydroxylation reaction in the degradation of nicotinate by aerobic bacteria. While the structure and critical residues involved in catalysis have been reported, the mechanism of this multistep enzyme has yet to be determined. A kinetic understanding of the NicC mechanism would enable comparison to other phenolic hydroxylases and illuminate its bioengineering potential for remediation of N-heterocyclic aromatic compounds. Toward these goals, transient state kinetic analyses by stopped-flow spectrophotometry were utilized to follow rapid changes in flavoenzyme absorbance spectra during all three stages of NicC catalysis: (1) 6-HNA binding; (2) NADH binding and FAD reduction; and (3) O2 binding with C4a-adduct formation, substrate hydroxylation, and FAD regeneration. Global kinetic simulations by numeric integration were used to supplement analytical fitting of time-resolved data and establish a kinetic mechanism. Results indicate that 6-HNA binding is a two-step process that substantially increases the affinity of NicC for NADH and enables the formation of a charge-transfer-complex intermediate to enhance the rate of flavin reduction. Singular value decomposition of the time-resolved spectra during the reaction of the substrate-bound, reduced enzyme with dioxygen provides evidence for the involvement of C4a-hydroperoxy-flavin and C4a-hydroxy-flavin intermediates in NicC catalysis. Global analysis of the full kinetic mechanism suggests that steady-state catalytic turnover is partially limited by substrate hydroxylation and C4a-hydroxy-flavin dehydration to regenerate the flavoenzyme. Insights gleaned from the kinetic model and determined microscopic rate constants provide a fundamental basis for understanding NicC's substrate specificity and reactivity.

Crystal structure of MbnF: an NADPH-dependent flavin monooxygenase from Methylocystis strain SB2.

Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X-Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.

Uncovering Zn2+ as a cofactor of FAD-dependent Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate dehydrogenase

Pseudomonas aeruginosa couples the oxidation of d-2-hydroxyglutarate (D2HG) to l-serine biosynthesis for survival, using d-2-hydroxyglutarate dehydrogenase from P.aeruginosa (PaD2HGDH). Knockout of PaD2HGDH impedes P.aeruginosa growth, making PaD2HGDH a potential target for therapeutics. Previous studies showed that the enzyme's activity increased with Zn2+, Co2+, or Mn2+ but did not establish the enzyme's metal composition and whether the metal is an activator or a required cofactor for the enzyme, which we addressed in this study. Comparable to the human enzyme, PaD2HGDH showed only 15% flavin reduction with D2HG or d-malate. Upon purifying PaD2HGDH with 1mM Zn2+, the Zn2+:protein stoichiometry was 2:1, yielding an enzyme with ∼40s-1kcat for d-malate. Treatment with 1mM EDTA decreased the Zn2+:protein ratio to 1:1 without changing the kinetic parameters with d-malate. We observed complete enzyme inactivation for the metalloapoenzyme with 100mM EDTA treatment, suggesting that Zn2+ is essential for PaD2HGDH activity. The presence of Zn2+ increased the flavin N3 atom pKa value to 11.9, decreased the flavin ε450 at pH 7.4 from 13.5 to 11.8 mM-1cm-1, and yielded a charged transfer complex with a broad absorbance band >550nm, consistent with a Zn2+-hydrate species altering the electronic properties of the enzyme-bound FAD. The exogenous addition of Zn2+, Co2+, Cd2+, Mn2+, or Ni2+ to the metalloapoenzyme reactivated the enzyme in a sigmoidal pattern, consistent with an induced fit rapid-rearrangement mechanism. Collectively, our data demonstrate that PaD2HGDH is a Zn2+-dependent metallo flavoprotein, which requires Zn2+ as an essential cofactor for enzyme activity.

The Pseudomonas aeruginosa PAO1 metallo flavoprotein d-2-hydroxyglutarate dehydrogenase requires Zn2+ for substrate orientation and activation

Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate (D2HG) dehydrogenase (PaD2HGDH) oxidizes D2HG to 2-ketoglutarate during the vital l-serine biosynthesis and is a potential therapeutic target against P.aeruginosa. PaD2HGDH, which oxidizes d-malate as an alternative substrate, has been demonstrated to be a metallo flavoprotein that requires Zn2+ for activity. However, the role of Zn2+ in the enzyme has not been elucidated, making it difficult to rationalize why nature employs both a redox center and a metal ion for catalysis in PaD2HGDH and other metallo flavoenzymes. In this study, recombinant His-tagged PaD2HGDH was purified to high levels in the presence of Zn2+ or Co2+ to investigate the metal's role in catalysis. We found that the flavin reduction step was reversible and partially rate limiting for the enzyme's turnover at pH 7.4 with either D2HG or d-malate with similar rate constants for both substrates, irrespective of whether Zn2+ or Co2+ was bound to the enzyme. The steady-state pL profiles of the kcat and kcat/Km values with d-malate demonstrate that Zn2+ mediates the activation of water coordinated to the metal. Our data are consistent with a dual role for the metal, which orients the hydroxy acid substrate in the enzyme's active site and rapidly deprotonates the substrate to yield an alkoxide species for hydride transfer to the flavin. Thus, we propose a catalytic mechanism for PaD2HGDH oxidation that establishes Zn2+ as a cofactor required for substrate orientation and activation during enzymatic turnover.

A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions.

Many bacteria of the genus Shewanella are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments. The Mtr complex is expressed under anoxic and oxygen-limited conditions and contains an extracellular MtrC subunit. This has a conserved CX8C motif that inhibits aerobic growth when removed. This inhibition is caused by an increase in ROS that kills the majority of S. oneidensis cells in culture. To better understand this effect, soluble MtrC isoforms with modified CX8C were isolated. These isoforms produced increased concentrations of H2O2 in the presence of flavin mononucleotide (FMN) and greatly increased the affinity between MtrC and FMN. X-ray crystallography revealed that the molecular structure of MtrC isoforms was largely unchanged, while small-angle X-ray scattering suggested that a change in flexibility was responsible for controlling FMN binding. Together, these results reveal that FMN reduction in S. oneidensis MR-1 is controlled by the redox-active disulfide on the cytochrome surface. In the presence of oxygen, the disulfide forms, lowering the affinity for FMN and decreasing the rate of peroxide formation. This cysteine pair consequently allows the cell to respond to changes in oxygen level and survive in a rapidly transitioning environment. IMPORTANCE Bacteria that live at the oxic/anoxic interface have to rapidly adapt to changes in oxygen levels within their environment. The facultative anaerobe Shewanella oneidensis MR-1 can use EET to respire in the absence of oxygen, but on exposure to oxygen, EET could directly reduce extracellular oxygen and generate harmful reactive oxygen species that damage the bacterium. By modifying an extracellular cytochrome called MtrC, we show how preventing a redox-active disulfide from forming causes the production of cytotoxic concentrations of peroxide. The disulfide affects the affinity of MtrC for the redox-active flavin mononucleotide, which is part of the EET pathway. Our results demonstrate how a cysteine pair exposed on the surface controls the path of electron transfer, allowing facultative anaerobic bacteria to rapidly adapt to changes in oxygen concentration at the oxic/anoxic interface.

An RNA aptamer that shifts the reduction potential of metabolic cofactors

The discovery of ribozymes has inspired exploration of RNA’s potential to serve as primordial catalysts in a hypothesized RNA world. Modern oxidoreductase enzymes employ differential binding between reduced and oxidized forms of redox cofactors to alter cofactor reduction potential and enhance the enzyme’s catalytic capabilities. The utility of differential affinity has been underexplored as a chemical strategy for RNA. Here we show an RNA aptamer that preferentially binds oxidized forms of flavin over reduced forms and markedly shifts flavin reduction potential by −40 mV, similar to shifts for oxidoreductases. Nuclear magnetic resonance structural analysis revealed π–π and donor atom–π interactions between the aptamer and flavin that cause unfavorable contacts with the electron-rich reduced form, suggesting a mechanism by which the local environment of the RNA-binding pocket drives the observed shift in cofactor reduction potential. It seems likely that primordial RNAs could have used similar strategies in RNA world metabolisms.

Kinetic solvent viscosity effects uncover an internal isomerization of the enzyme-substrate complex in Pseudomonas aeruginosa PAO1 NADH:Quinone oxidoreductase

NAD(P)H:quinone oxidoreductases (NQOs) play an essential protective role as antioxidants in the detoxification of quinones in both Prokaryotes and Eukaryotes. NQO from Pseudomonas aeruginosa PAO1 uses FMN to catalyze the two-electron reduction of various quinones with NADH. In this study, steady-state kinetics, kinetic solvent viscosity effects, and rapid reaction kinetics were used to determine which kinetic steps control the overall turnover of the enzyme with benzoquinone or juglone. The rate constant for flavin reduction (kred) at pH 6.0 was 12.9 ± 0.3 s−1, and the Kd for NADH was at least an order of magnitude lower than 90 μM. With benzoquinone, the kcat value was 11.7 ± 0.3 s−1, consistent with flavin reduction being almost entirely rate-limiting for overall turnover. With juglone, a kcat value of 10.0 ± 0.5 s−1 was recorded. The normalized plot of the relative solvent viscosity effects on the kcat values established that hydride transfer from NADH to the FMN and quinol product release, with a calculated rate constant (kP-rel) of 52 s−1, are partially rate-limiting for the overall turnover of NQO. Kinetic solvent viscosity effects with glucose or sucrose revealed a hyperbolic dependence on the kcat and kcat/Km values with benzoquinone or juglone, respectively, consistent with the presence of a solvent-sensitive internal isomerization of the enzyme-substrate complex (ES). The data demonstrate opposing effects of benzoquinone and juglone on the equilibrium of the NQO ES isomerization with glucose or sucrose. Thus, our study demonstrates how quinol substrate properties alter the equilibrium of NQO ES isomerization.

H2-Driven Reduction of Flavin by Hydrogenase Enables Cleaner Operation of Nitroreductases for Nitro-Group to Amine Reductions

Hydrogenase-mediated reduction of flavin mononucleotide by H2 is exploited to enable cleaner application of nitroreductase enzymes for reduction of aromatic nitro functional groups. This turns the overall reaction into a biocatalytic hydrogenation. Use of flavin-containing nitroreductases in industrial biotechnology typically relies upon NADH or NADPH as reductant, together with glucose dehydrogenase and glucose as a regeneration system for the reduced nicotinamide cofactor, with 3 equivalents of the carbon-intensive glucose required for a single 6-electron nitro to amine conversion. We show here that reduced flavin mononucleotide is an alternative reductant for nitroreductases, and by combining this with H2-driven recycling of reduced flavin, we avoid glucose, thereby enabling atom-efficient biocatalytic nitro reductions. We compare this biocatalytic system, via green chemistry metrics, to existing strategies for biocatalytic nitro-group reductions, particularly with respect to replacing glucose with H2 gas. We take steps towards demonstrating industrial viability: we report an overexpression system for E. coli hydrogenase 1, giving a 12-fold improvement in enzyme yield; we show a reaction in which the hydrogenase exhibits > 26,000 enzyme turnovers; and we demonstrate reasonable solvent tolerance of the hydrogenase and flavin reduction system which would enable reaction intensification.

Investigating Substrate Specificity For 6‐Hydroxynicotinate 3‐Monooxygenase (NicC) With Coumalic Acid: Consequences To Catalysis Of Replacing or Removing The Ring Nitrogen

6‐Hydroxynicotinate 3‐monooxygenase (NicC) is a group A flavin dependent monooxygenase (FDM) that catalyzes the regioselective decarboxylative hydroxylation of 6‐HNA into 2,5‐dihydroxypyridine (DHP) with the concomitant oxidation of NADH to NAD+. This enzyme‐catalyzed reaction involves three segments: (1) substrate binding, (2) FAD reduction by NADH, and (3) hydroxylation‐decarboxylation of 6‐HNA through a hydroperoxy‐flavin intermediate. Past studies have shown that NicC can catalyze this reaction with alternate aromatic substrates including 4‐hydroxybenzoic acid (4‐HBA), a substrate analogue with a homocyclic ring (i.e. lacking the ring nitrogen). However, 4‐HBA is bound weakly (355 ± 66 μM) and only 50% of the reaction progresses to substrate hydroxylation, versus hydroperoxy‐flavin degradation to H2O2, suggesting the importance of the ring nitrogen in the coupling efficiency of NicC. To further explore the role of the ring nitrogen, the efficiency of the reaction with coumalic acid (CA) was determined. The NicC‐CA dissociation constant measured by monitoring the change in absorbance at 450nm (flavin) shows that NicC binds CA (Kd = 5 ± 1 μM) 10x more tightly than 6‐HNA (Kd = 58 ± 12 μM). Binding of CA by NicC is an isothermal process (∆H = 0), indicating that it is driven by a favorable change in entropy. Analysis of the reaction products by Q‐TOF MS indicates that NicC catalyzes the decarboxylative hydroxylation of CA into 5‐hydroxy‐2H‐pyrane‐2‐one. However, coupling experiments by HPLC analysis and detection of H2O2 by a fluorescence assay both indicate that the reaction with CA is highly uncoupled with only minor 5‐hydroxy‐2H‐pyrane‐2‐one formation. Steady‐state kinetic analyses show that CA bound to NicC enhances its rate of NADH oxidation 16‐fold (by comparison, 6‐HNA enhances the rate of NicC NADH oxidation 30‐fold). Transient state kinetic analyses by stopped flow under anaerobic conditions to permit single‐turnover kinetics shows that, unlike in the binding of 6‐HNA, the binding of CA does not enable the formation of a charge‐transfer complex prior to FAD reduction. These results indicate that an H‐bond accepting group in the aromatic ring at position 1 is important for establishing the correct conformation of the ES complex to enable effective binding, flavin reduction and, ultimately, substrate hydroxylation.

Exploring The Promiscuity Potential Of 6‐Hydroxynicotinate‐3‐Monooxygenase: Consequences To Catalysis Of Adding A Nitrogen At C5 Within The Substrate’s Aromatic Ring

Bacterial degradation of nicotinic acid is a model for exploring the bioremediation potential of soil bacteria. One enzyme within this catabolic pathway is 6‐hydroxynicotinate‐3‐monooxygenase (NicC), a group A flavoenzyme that catalyzes the conversion of 6‐hydroxynicotinate (6HNA) to 2,5‐dihydroxypyridine. This decarboxylative hydroxylation reaction is relatively unique in biology. We recently published a study highlighting NicC’s ability to catalyze the decarboxylation and hydroxylation reaction with alternative substrates including 4‐hydroxybenzoate (4HBA) and 5‐chloro‐6‐hydroxynicotinate (5‐Cl‐6HNA). To further explore the structural basis of a substrate that enables tight binding and catalysis with NicC, 2‐hydroxypyrimidine‐5‐caboxylate (2HM5A) was characterized. This 6‐HNA analogue with an extra N atom at C5 was chosen due to its fundamental symmetry and comparable electron distribution as 5‐Cl‐6HNA. Analyses of NicC with 2HM5A show that the expected product, 2,5‐dihydroxypyrimidine (isouracil), is generated, with H2O2 occurring at a mol fraction of about 0.07, indicating that uncoupling between NADH oxidation and substrate hydroxylation is minimal. Equilibrium titration of NicC with 2HM5A by changes in A‐450 nm measure the Kd = 39 ± 6 µM, indicating that this substrate is bound more tightly than 6‐HNA (Kd= 58 ± 12 µM) but less tightly than 5‐Cl‐6‐HNA (Kd= 7 ± 2 µM). Interestingly, the overall ΔA450 elicited by the binding of 2HM5A (0.018) was smaller in comparison to the ΔA450 observed for the binding of 5‐Cl‐6HNA (0.07) or 6‐HNA (0.05). Attenuated flavin movement due the extra nitrogen of the pyrimidine ring in 2HM5A H‐bonding to the isoalloxazine ring may explain this observation. The kinetics of binding 2HM5A by NicC measured by stopped flow spectrophotometry shows that this analogue binds by a two‐step mechanism, with a nearly ten‐fold higher initial affinity for NicC prior to isomerization (ionization) than 6‐HNA. This added affinity may again be explainable by the added H‐bonding potential of this analogue. In the second step of binding, 2HM5A was observed to have a rate constant similar to 5‐Cl‐6HNA (k2= 220 s‐1), possibly indicating that increased electron density at/near the C5 position accelerates this phase of the binding mechanism. How 2HM5A affects NicC’s mechanism of flavin reduction and oxidation will also be presented. These findings provide further evidence of this monooxygenase’s potential to be engineered to hydroxylate other N‐heterocyclic aromatic compounds in bioremediation.