An active site mutation induces oxygen reactivity in D-arginine dehydrogenase: A case of superoxide diverting protons.

Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O2) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E246) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E246 residue with leucine affects PaDADH catalysis and its ability to react with O2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O2 reactivity in the E246L variant, resulting in a reduced flavin semiquinone species and superoxide (O2•-) during substrate oxidation. The O2•- reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme's log (kcat/Km) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O2•- can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins.

Targeted Mutation of a Non-catalytic Gating Residue Increases the Rate of Pseudomonas aeruginosa d-Arginine Dehydrogenase Catalytic Turnover.

Commercial food and l-amino acid industries rely on bioengineered d-amino acid oxidizing enzymes to detect and remove d-amino acid contaminants. However, the bioengineering of enzymes to generate faster biological catalysts has proven difficult as a result of the failure to target specific kinetic steps that limit enzyme turnover, kcat, and the poor understanding of loop dynamics critical for catalysis. Pseudomonas aeruginosa d-arginine dehydrogenase (PaDADH) oxidizes most d-amino acids and is a good candidate for application in the l-amino acid and food industries. The side chain of the loop L2 E246 residue located at the entrance of the PaDADH active site pocket potentially favors the closed active site conformation and secures the substrate upon binding. This study used site-directed mutagenesis, steady-state, and rapid reaction kinetics to generate the glutamine, glycine, and leucine variants and investigate whether increasing the rate of product release could translate to an increased enzyme turnover rate. Upon E246 mutation to glycine, there was an increased rate of d-arginine turnover kcat from 122 to 500 s-1. Likewise, the kcat values increased 2-fold for the glutamine or leucine variants. Thus, we have engineered a faster biocatalyst for industrial applications by selectively increasing the rate of the PaDADH product release.

Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins.

iLOV is an engineered flavin-binding fluorescent protein (FbFP) with applications for in vivo cellular imaging. To expand the range of applications of FbFPs for multicolor imaging and FRET-based biosensing, it is desirable to understand how to modify their absorption and emission wavelengths (i.e., through spectral tuning). There is particular interest in developing FbFPs that absorb and emit light at longer wavelengths, which has proven challenging thus far. Existing spectral tuning strategies that do not involve chemical modification of the flavin cofactor have focused on placing positively charged amino acids near flavin's C4a and N5 atoms. Guided by previously reported electrostatic spectral tunning maps (ESTMs) of the flavin cofactor and by quantum mechanical/molecular mechanical (QM/MM) calculations reported in this work, we suggest an alternative strategy: placing a negatively charged amino acid near flavin's N1 atom. We predict that a single-point mutant, iLOV-Q430E, has a slightly red-shifted absorption and fluorescence maximum wavelength relative to iLOV. To validate our theoretical prediction, we experimentally expressed and purified iLOV-Q430E and measured its spectral properties. We found that the Q430E mutation results in a slight change in absorption and a 4-8 nm red shift in the fluorescence relative to iLOV, in good agreement with the computational predictions. Molecular dynamics simulations showed that the carboxylate side chain of the glutamate in iLOV-Q430E points away from the flavin cofactor, which leads to a future expectation that further red shifting may be achieved by bringing the side chain closer to the cofactor.

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 1 mM Zn2+, the Zn2+:protein stoichiometry was 2:1, yielding an enzyme with ∼40 s-1kcat for d-malate. Treatment with 1 mM 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 100 mM 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-1 cm-1, and yielded a charged transfer complex with a broad absorbance band >550 nm, 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.

Non-active Site Residue in Loop L4 Alters Substrate Capture and Product Release in d-Arginine Dehydrogenase.

Numerous studies demonstrate that enzymes undergo multiple conformational changes during catalysis. The malleability of enzymes forms the basis for allosteric regulation: residues located far from the active site can exert long-range dynamical effects on the active site residues to modulate catalysis. The structure of Pseudomonas aeruginosa d-arginine dehydrogenase (PaDADH) shows four loops (L1, L2, L3, and L4) that span the substrate and the FAD-binding domains. Loop L4 comprises residues 329-336, spanning over the flavin cofactor. The I335 residue on loop L4 is ∼10 Šaway from the active site and ∼3.8 Šfrom N(1)-C(2)═O atoms of the flavin. In this study, we used molecular dynamics and biochemical techniques to investigate the effect of the mutation of I335 to histidine on the catalytic function of PaDADH. Molecular dynamics showed that the conformational dynamics of PaDADH are shifted to a more closed conformation in the I335H variant. In agreement with an enzyme that samples more in a closed conformation, the kinetic data of the I335H variant showed a 40-fold decrease in the rate constant of substrate association (k1), a 340-fold reduction in the rate constant of substrate dissociation from the enzyme-substrate complex (k2), and a 24-fold decrease in the rate constant of product release (k5), compared to that of the wild-type. Surprisingly, the kinetic data are consistent with the mutation having a negligible effect on the reactivity of the flavin. Altogether, the data indicate that the residue at position 335 has a long-range dynamical effect on the catalytic function in PaDADH.

Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1.

Enzymes require flexible regions to adopt multiple conformations during catalysis. The mobile regions of enzymes include gates that modulate the passage of molecules in and out of the enzyme's active site. The enzyme PA1024 from Pseudomonas aeruginosa PA01 is a recently discovered flavin-dependent NADH:quinone oxidoreductase (NQO, EC 1.6.5.9). Q80 in loop 3 (residues 75-86) of NQO is ∼15 Å away from the flavin and creates a gate that seals the active site through a hydrogen bond with Y261 upon NADH binding. In this study, we mutated Q80 to glycine, leucine, or glutamate to investigate the mechanistic significance of distal residue Q80 in NADH binding in the active site of NQO. The UV-visible absorption spectrum reveals that the mutation of Q80 minimally affects the protein microenvironment surrounding the flavin. The anaerobic reductive half-reaction of the NQO-mutants yields a ≥25-fold increase in the Kd value for NADH compared to the WT enzyme. However, we determined that the kred value was similar in the Q80G, Q80L, and wildtype enzymes and only ∼25% smaller in the Q80E enzyme. Steady-state kinetics with NQO-mutants and NQO-WT at varying concentrations of NADH and 1,4-benzoquinone establish a ≤5-fold decrease in the kcat/KNADH value. Moreover, there is no significant difference in the kcat/KBQ (∼1 × 106 M-1s-1) and kcat (∼24 s-1) values in NQO-mutants and NQO-WT. These results are consistent with the distal residue Q80 being mechanistically essential for NADH binding to NQO with minimal effect on the quinone binding to the enzyme and hydride transfer from NADH to flavin.

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.

Discovery of a new flavin N5-adduct in a tyrosine to phenylalanine variant of d-Arginine dehydrogenase.

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) catalyzes the flavin-dependent oxidation of d-arginine and other d-amino acids. Here, we report the crystal structure at 1.29 Å resolution for PaDADH-Y249F expressed and co-crystallized with d-arginine. The overall structure of PaDADH-Y249F resembled PaDADH-WT, but the electron density for the flavin cofactor was ambiguous, suggesting the presence of modified flavins. Electron density maps and mass spectrometric analysis confirmed the presence of both N5-(4-guanidino-oxobutyl)-FAD and 6-OH-FAD in a single crystal of PaDADH-Y249F and helped with the further refinement of the X-ray crystal structure. The versatility of the reduced flavin is apparent in the PaDADH-Y249F structure and is evidenced by the multiple functions it can perform in the same active site.

Ionic Atmosphere Effect on the Absorption Spectrum of a Flavoprotein: A Reminder to Consider Solution Ions.

This study utilizes the FMN-dependent NADH:quinone oxidoreductase from Pseudomonas aeruginosa PAO1 to investigate the effect of introducing an active site negative charge on the flavin absorption spectrum both in the absence and presence of a long-range electrostatic potential coming from solution ions. There were no observed changes in the flavin UV-visible spectrum when an active site tyrosine (Y277) becomes deprotonated in vitro. These results could only be reproduced computationally using average solvent electrostatic configuration (ASEC) QM/MM simulations that include both positive and negative solution ions. The same calculations performed with minimal ions to neutralize the total protein charge predicted that deprotonating Y277 would significantly alter the flavin absorption spectrum. Analyzing the distribution of solution ions indicated that the ions reorganize around the protein surface upon Y277 deprotonation to cancel the effect of the tyrosinate on the flavin absorption spectrum. Additional biochemical experiments were performed to test this hypothesis.

Tuning Protein Dynamics to Sense Rapid Endoplasmic-Reticulum Calcium Dynamics.

Multi-scale calcium (Ca2+ ) dynamics, exhibiting wide-ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic-reticulum (ER)-targeted Ca2+ indicator, R-CatchER, which showed superior kinetics in vitro (koff ≥2×103  s-1 , kon ≥7×106  M-1 s-1 ) and in multiple cell types. R-CatchER captured spatiotemporal ER Ca2+ dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose a principle to rationally design genetically encoded Ca2+ indicators with a single Ca2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore. The design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range. Our work may facilitate protein design, visualizing Ca2+ dynamics, and drug discovery.

Rapid subcellular calcium responses and dynamics by calcium sensor G-CatchER.

The precise spatiotemporal characteristics of subcellular calcium (Ca2+) transients are critical for the physiological processes. Here we report a green Ca2+ sensor called "G-CatchER+" using a protein design to report rapid local ER Ca2+ dynamics with significantly improved folding properties. G-CatchER+ exhibits a superior Ca2+ on rate to G-CEPIA1er and has a Ca2+-induced fluorescence lifetimes increase. G-CatchER+ also reports agonist/antagonist triggered Ca2+ dynamics in several cell types including primary neurons that are orchestrated by IP3Rs, RyRs, and SERCAs with an ability to differentiate expression. Upon localization to the lumen of the RyR channel (G-CatchER+-JP45), we report a rapid local Ca2+ release that is likely due to calsequestrin. Transgenic expression of G-CatchER+ in Drosophila muscle demonstrates its utility as an in vivo reporter of stimulus-evoked SR local Ca2+ dynamics. G-CatchER+ will be an invaluable tool to examine local ER/SR Ca2+ dynamics and facilitate drug development associated with ER dysfunction.

A Single-Point Mutation in d-Arginine Dehydrogenase Unlocks a Transient Conformational State Resulting in Altered Cofactor Reactivity.

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.

Kinetic and Bioinformatic Characterization of d-2-Hydroxyglutarate Dehydrogenase from Pseudomonas aeruginosa PAO1.

d-2-Hydroxyglutarate dehydrogenase from Pseudomonas aeruginosa PAO1 (PaD2HGDH) catalyzes the oxidation of d-2-hydroxyglutarate to 2-ketoglutarate, which is a necessary step in the serine biosynthetic pathway. The dependence of P. aeruginosa on PaD2HGDH makes the enzyme a potential therapeutic target against P. aeruginosa. In this study, recombinant His-tagged PaD2HGDH was expressed and purified to high levels from gene PA0317, which was previously annotated as an FAD-binding PCMH-type domain-containing protein. The enzyme cofactor was identified as FAD with fluorescence emission after phosphodiesterase treatment and with mass spectrometry analysis. PaD2HGDH had a kcat value of 11 s-1 and a Km value of 60 µM with d-2-hydroxyglutarate at pH 7.4 and 25 °C. The enzyme was also active with d-malate but did not react with molecular oxygen. Steady-state kinetics with d-malate and phenazine methosulfate as an electron acceptor established a mechanism that was consistent with ping-pong bi-bi steady-state kinetics at pH 7.4. A comparison of the kcat/Km values with d-2-hydroxyglutarate and d-malate suggested that the C5 carboxylate of d-2-hydroxyglutarate is important for the substrate specificity of the enzyme. Other homologues of the enzyme have been previously grouped in the VAO/PMCH family of flavoproteins. PaD2HGDH shares fully conserved residues with other α-hydroxy acid oxidizing enzymes, and these conserved residues are found in the active site of the PaD2HDGH homology model. An Enzyme Function Initiative-Enzyme Similarity Tool Sequence Similarity Network analysis suggests a functional difference between PaD2HGDH and human D2HGDH, and no relationship with VAO. A phylogenetic tree analysis of PaD2HGDH, VAO, and human D2HGDH establishes genetic diversity among these enzymes.

Kinetic solvent viscosity effects reveal a protein isomerization in the reductive half-reaction of Neurospora crassa class II nitronate monooxygenase.

Nitronate monooxygenase from Neurospora crassa (NcNMO) is an FMN-dependent enzyme that oxidizes nitronates. The enzyme belongs to Group H of flavin monooxygenases. Previous biochemical and mechanistic studies on the enzyme showed that NcNMO oxidizes both anionic nitronates and neutral nitroalkanes, a feature distinguishing NcNMO from bacterial and other fungal NMOs which are active exclusively on nitronates. Recently, NMOs have been shown to oxidize propionate 3-nitronate (P3N), a toxic nitro acid present in legumes, fungi, and leaf beetles; P3N is an irreversible inhibitor of succinate dehydrogenase causing anywhere from neurological disorders to death in livestock and humans. In this study, we report the first kinetic investigation of NcNMO with P3N and its conjugated acid 3-nitropropionic acid (3NPA), and a mechanistic investigation with 3NPA using kinetic solvent viscosity effects. The kcat value with P3N (300 s-1) was 7-times larger than with 3NPA and the kcat/KP3N value (1,700,000 M-1s-1) was ~500-times larger than the kcat/K3NPA value, consistent with P3N being a faster and better substrate than 3NPA. The normalized kcat and kcat/K3NPA values showed inverse hyperbolic dependences on the relative solvent viscosity, consistent with an internal isomerization of the enzyme-substrate complex. A similar inverse hyperbolic pattern of the normalized kred value for the rate constant of flavin reduction determined anaerobically in a stopped-flow spectrophotometer suggested that the internal enzyme isomerization occurs before the flavin reduction step in the reductive half-reaction.

Choline oxidases.

Choline oxidase catalyzes the four-electron, two-step, flavin-mediated oxidation of choline to glycine betaine. The enzyme is important both for medical and biotechnological reasons, because glycine betaine is one among a limited number of compatible solutes used by cells to counteract osmotic pressure. From a fundamental standpoint, choline oxidase has emerged as one of the paradigm enzymes for the oxidation of alcohols catalyzed by flavoproteins. Mechanistic, structural, and computational studies have elucidated the mechanism of action of the enzyme from Arthrobacter globiformis at the molecular level. Both choline and oxygen access to the active site cavity are gated and tightly controlled. Amino acid residues involved in substrate binding, and their contribution, have been identified. The mechanism of choline oxidation, with a hydride transfer reaction, an asynchronous transition state, the formation and stabilization of an alkoxide transient species, and a quantum mechanical mode of reaction, has been elucidated. The importance of nonpolar side chains for oxygen localization and of the positive charge harbored on the substrate for activation of oxygen for reaction with the reduced flavin have been recognized. Interesting phenomena, like the formation of a metastable photoinduced flavin-protein adduct, the reversible formation of a bicovalent flavoprotein, and the trapping of the enzyme in inactive conformations, have been described. This review summarizes the current status of our understanding on the structure-function-dynamics of choline oxidase.

A Metastable Photoinduced Protein-Flavin Adduct in Choline Oxidase, an Enzyme Not Involved in Light-Dependent Processes.

Photoinduced formation of protein-flavin adducts is crucial in photoresponsive proteins containing light-oxygen-voltage (LOV) domains. LOV proteins typically share an N-terminal sensor domain with FMN and a C-terminal effector domain such as a kinase, a phosphodiesterase, or a DNA-binding protein. Light absorption by FMN results in a covalent flavin-cysteine adduct, which allosterically translates to the linked effector domain. Photoinduced protein-flavin adducts have not been reported in enzymes not involved in light-dependent processes. Here, we have used fluorescence, pH effects, and mutagenesis to follow up a serendipitous observation of an unusual fluorescence excitation spectrum in choline oxidase at alkaline pH (M. Ghanem and G. Gadda, unpublished observations). Physiologically, choline oxidase oxidizes choline to betaine through two FAD-associated reactions and is not a photoenzyme. The enzyme-bound flavin showed a progressive shift of the fluorescence excitation maximum (λex) from 468 to 399 nm with increasing pH values between pH 6.0 and 10.0, consistent with a metastable photoinduced protein-flavin adduct. In contrast, the maximal λem was independent of pH, with values of ∼526 nm. For comparison, fluorescence spectra of FAD in bulk solution had maximal values differing by ≤2 nm at different pH values, with λex at 453 nm and λem at 527 nm. The unusual behavior of the enzyme persisted in the mutated S101A enzyme variant but was eliminated in the H466Q variant, suggesting that the photoinduced species is likely a C4a-N-histidyl-FAD. The results provide evidence that metastable photoinduced formation of a flavin-protein adduct can occur in an enzyme that is not a photoreceptor or a photoenzyme.

Characterization of conserved active site residues in class I nitronate monooxygenase.

Propionate 3-nitronate (P3N) is a natural toxin that irreversibly inhibits mitochondrial succinate dehydrogenase. P3N poisoning leads to a variety of neurological disorders and even death. Nitronate monooxygenase (NMO) from Pseudomonas aeruginosa PAO1 was the first NMO characterized in bacteria and serves as a paradigm for Class I NMO. Here, we hypothesized that the carboxylate group of P3N might form a hydrogen bond with one or more of the four tyrosine or a lysine residues that are conserved in the active site of the enzyme. In the wild-type enzyme, the kcat value was pH independent between pH 6.0 and 11.0, while the kcat/KP3N value decreased at high pH, suggesting that a protonated group with a pKa value of 9.5 is required for binding the anionic substrate. A pH titration of the UV-visible absorption spectrum of the enzyme showed an increased absorbance at 297 nm with increasing pH, defining a pKa value of 9.5 and a Δε297 nm of 2.4 M-1cm-1, consistent with a tyrosine being important for substrate binding. The N3 atom of the oxidized flavin, instead, did not ionize likely because its pKa was perturbed by the ionization of a tyrosine in the active site of the enzyme. The Y109F, Y254F, Y299F, Y303F, and K307 M, substitutions had small effects (i.e., <3.5-fold) on the steady-state kinetic parameters of the enzyme. With all mutated enzymes, the kcat/KP3N value was less than 2.5-fold different from the wild-type enzyme, suggesting that none of the residues is solely essential for substrate binding.

On the use of noncompetitive kinetic isotope effects to investigate flavoenzyme mechanism.

This account describes the application of kinetic isotope effects (KIEs) to investigate the mechanistic properties of flavin dependent enzymes. Assays can be conducted during steady-state catalytic turnover of the flavoenzyme with its substrate or by using rapid-kinetic techniques to measure either the reductive or oxidative half-reactions of the enzyme. Great care should be taken to ensure that the observed effects are due to isotopic substitution and not other factors such as pH effects or changes in the solvent viscosity of the reaction mixture. Different types of KIEs are described along with a physical description of their origins and the unique information each can provide about the mechanism of an enzyme. Detailed experimental techniques are outlined with special emphasis on the proper controls and data analysis that must be carried out to avoid erroneous conclusions. Examples are provided for each type of KIE measurement from references in the literature. It is our hope that this article will clarify any confusion concerning the utility of KIEs in the study of flavoprotein mechanism and encourage their use by the community.

Kinetic Investigation of a Presumed Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes a New Class of NAD(P)H:Quinone Reductases.

PA0660 from Pseudomonas aeruginosa PAO1 is currently classified as a hypothetical nitronate monooxygenase (NMO), but no evidence at the transcript or protein level has been presented. In this study, PA0660 was purified and its biochemical and kinetic properties were characterized. Absorption spectroscopy and mass spectrometry demonstrated a tightly, noncovalently bound FMN in the active site of the enzyme. Analytical ultracentrifugation showed that the enzyme exists as a dimer in solution. Despite its annotation, PA0660 did not exhibit nitronate monooxygenase activity. The enzyme could be reduced with NADPH or NADH with a marked preference for NADPH, as indicated by ∼30-fold larger kcat/ Km and kred/ Kd values. Turnover could be sustained with NAD(P)H and quinones, DCPIP, and to a lesser extent molecular oxygen. However, PA0660 did not turn over with methyl red, consistent with a lack of azoreductase activity. The enzyme turned over through a ping-pong bi-bi steady-state kinetic mechanism with NADPH and 1,4-benzoquinone showing a kcat value of 90 s-1. The rate constant for flavin reduction with saturating NADPH was 360 s-1, whereas that for flavin oxidation with 1,4-benzoquinone was 270 s-1, consistent with both hydride transfers from the pyridine nucleotide to the flavin and from the flavin to 1,4-benzoquinone being partially rate-limiting for enzyme turnover. A BlastP search and a multiple-sequence alignment analysis of PA0660 highlighted the presence of six conserved motifs in >1000 open reading frames currently annotated as hypothetical NMOs. Our results suggest that PA0660 should be classified as an NAD(P)H:quinone reductase and serve as a paradigm enzyme for a new class of enzymes.