Investigations of two-component flavin-dependent monooxygenase systems.

Bacterial two-component flavin-dependent monooxygenase systems catalyze the oxidation of diverse metabolic reactions. There are several shared mechanistic features in the two-component monooxygenase systems that differ from canonical monooxygenase enzymes. The flavin reductases catalyze the reductive half-reaction, and the reduced flavin is transferred to the monooxygenase enzyme. The oxidative half-reaction catalyzed by the monooxygenase enzyme has been proposed to occur through the formation of a (hydro)peroxyflavin intermediate. In some two-component flavin-dependent systems the mechanism of flavin transfer involves protein-protein interactions between the flavin reductase and monooxygenase enzyme. Methods are presented that provide an alternative approach from flavin-bound monooxygenases to evaluate the kinetic properties and flavin transfer mechanism of the two-component flavin-dependent monooxygenase systems.

Insights into Thiotemplated Pyrrole Biosynthesis Gained from the Crystal Structure of Flavin-Dependent Oxidase in Complex with Carrier Protein.

Sequential enzymatic reactions on substrates tethered to carrier proteins (CPs) generate thiotemplated building blocks that are then delivered to nonribosomal peptide synthetases (NRPSs) to generate peptidic natural products. The underlying diversity of these thiotemplated building blocks is the principal driver of the chemical diversity of NRPS-derived natural products. Structural insights into recognition of CPs by tailoring enzymes that generate these building blocks are sparse. Here we present the crystal structure of a flavin-dependent prolyl oxidase that furnishes thiotemplated pyrrole as the product, in complex with its cognate CP in the holo and product-bound states. The thiotemplated pyrrole is an intermediate that is well-represented in natural product biosynthetic pathways. Our results delineate the interactions between the CP and the oxidase while also providing insights into the stereospecificity of the enzymatic oxidation of the prolyl heterocycle to the aromatic pyrrole. Biochemical validation of the interaction between the CP and the oxidase demonstrates that NRPSs recognize and bind to their CPs using interactions quite different from those of fatty acid and polyketide biosynthetic enzymes. Our results posit that structural diversity in natural product biosynthesis can be, and is, derived from subtle modifications of primary metabolic enzymes.

Photoirradiation Generates an Ultrastable 8-Formyl FAD Semiquinone Radical with Unusual Properties in Formate Oxidase.

Formate oxidase (FOX) was previously shown to contain a noncovalently bound 8-formyl FAD (8-fFAD) cofactor. However, both the absorption spectra and the kinetic parameters previously reported for FOX are inconsistent with more recent reports. The ultraviolet-visible (UV-vis) absorption spectrum reported in early studies closely resembles the spectra observed for protein-bound 8-formyl flavin semiquinone species, thus suggesting FOX may be photosensitive. Therefore, the properties of dark and light-exposed FOX were investigated using steady-state kinetics and site-directed mutagenesis analysis along with inductively coupled plasma optical emission spectroscopy, UV-vis absorption spectroscopy, circular dichroism spectroscopy, liquid chromatography and mass spectrometry, and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, these experimental results demonstrate that FOX is deactivated in the presence of light through generation of an oxygen stable, anionic (red) 8-fFAD semiquinone radical capable of persisting either in an aerobic environment for multiple weeks or in the presence of a strong reducing agent like sodium dithionite. Herein, we study the photoinduced formation of the 8-fFAD semiquinone radical in FOX and report the first EPR spectrum of this radical species. The stability of the 8-fFAD semiquinone radical suggests FOX to be a model enzyme for probing the structural and mechanistic features involved in stabilizing flavin semiquinone radicals. It is likely that the photoinduced formation of a stable 8-fFAD semiquinone radical is a defining characteristic of 8-formyl flavin-dependent enzymes. Additionally, a better understanding of the radical stabilization process may yield a FOX enzyme with more robust activity and broader industrial usefulness.

A high-throughput pH-based colorimetric assay: application focus on alpha/beta hydrolases.

Research involving α/ß hydrolases, including α-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting α/ß hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a 'hit' or a 'miss,' in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of α/ß hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.

Mechanistic studies of formate oxidase from Aspergillus oryzae: A novel member of the glucose-Methanol-choline oxidoreductase enzyme superfamily that oxidizes carbon acids.

Formate oxidase (FOX) from Aspergillus oryzae is the only GMC member that oxidizes a carbon acid rather than alcohols; thus, its catalytic mechanism may be different from that of other GMC members. We have used pH, solvent viscosity, and deuterium kinetic isotope effects, to investigate the catalytic mechanism of FOX. The enzyme followed a Bi-Bi sequential steady-state kinetic mechanism. The kcat value was pH-independent between pH 2.8 and 6.8, suggesting a lack of ionizable groups in kinetic step(s) that limit the overall turnover of the enzyme. The kcat/Kformate value decreased from a value of 10,000 M-1s-1 at low pH with a pKa value of 4.4, consistent with the requirement of a protonated group for substrate binding. An inverse viscosity dependence on the kcat/Kformate value indicated an isomerization of the Michaelis complex. The kcat/Koxygen value was 340,000 M-1s-1 and pH independent up to pH 6.0. The Dkcat and D(kcat/Kformate) values were 2.5 and 1.9, respectively, indicating that substrate CH bond cleavage is rate-limiting for FOX catalysis. Analytical ultracentrifugation indicated a concentration dependence of the oligomeric state of FOX. The appkred,H value was ∼75% that of kcat,H, indicating that the anaerobic reduction of FOX was dependent on the oligomeric state of FOX.

Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties.

Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.

Steady-state kinetic isotope effects support a complex role of Arg226 in the proposed desulfonation mechanism of alkanesulfonate monooxygenase.

The alkanesulfonate monooxygenase system catalyzes the desulfonation of alkanesulfonates through proposed acid-base mechanistic steps that involves the abstraction of a proton from the alkane peroxyflavin intermediate and protonation of the FMN-O(-) intermediate. Both solvent and kinetic isotope studies were performed to define the proton transfer steps involved in the SsuD reaction. Substitution of the protium at the C1 position of octanesulfonate with deuterium resulted in an observed primary isotope effect of 3.0 ± 0.2 on the kcat parameter, supporting abstraction of the α-proton from the alkane peroxyflavin as the rate-limiting step in catalysis. Previous studies implicated Arg226 as the acid involved in the reprotonation of the hydroxyflavin intermediate. Solvent isotope kinetic studies gave an inverse isotope effect on (D2O)kcat of 0.75 ± 0.04 with no observable effect on (D2O)kcat/Km. This resulted in equivalent solvent isotope effects on (D2O)kcat and (D2O)(kcat)D, suggesting a solvent equilibrium isotope effect on a step occurring after the first irreversible step through product release. Data from proton inventory studies on kcat were best fit to a dome-shaped curve consistent with a conformational change to an open conformation during product release. The solvent isotope effect data coupled with the corresponding proton inventory results support and extend our previous observations that Arg226 donates a proton to the FMN-O(-) intermediate, triggering a conformational change that opens the enzyme to solvation and promotes product release.

Identification of critical steps governing the two-component alkanesulfonate monooxygenase catalytic mechanism.

The alkanesulfonate monooxygenase enzyme (SsuD) catalyzes the oxygenolytic cleavage of a carbon-sulfur bond from sulfonated substrates. A mechanism involving acid-base catalysis has been proposed for the desulfonation mechanism by SsuD. In the proposed mechanism, base catalysis is involved in abstracting a proton from the alkane peroxyflavin intermediate, while acid catalysis is needed for the protonation of the FMNO(-) intermediate. The pH profiles of k(cat) indicate that catalysis by SsuD requires a group with a pK(a) of 6.6 ± 0.2 to be deprotonated and a second group with a pK(a) of 9.5 ± 0.1 to be protonated. The upper pK(a) value was not present in the pH profiles of k(cat)/K(m). Several conserved amino acid residues (His228, His11, His333, Cys54, and Arg226) have been identified as having potential catalytic importance due to the similar spatial arrangements with close structural and functional relatives of SsuD. Substitutions to these amino acid residues were generated, and the pH dependencies were evaluated and compared to wild-type SsuD. Although a histidine residue was previously proposed to be the active site base, the His variants possessed similar steady-state kinetic parameters as wild-type SsuD. Interestingly, R226A and R226K SsuD variants possessed undetectable activity, and there was no detectable formation of the C4a-(hydro)peroxyflavin intermediate for the Arg226 SsuD variants. Guanidinium rescue with the R226A SsuD variant resulted in the recovery of 1.5% of the wild-type SsuD k(cat) value. These results implicate Arg226 playing a critical role in catalysis and provide essential insights into the mechanistic steps that guide the SsuD desulfonation process.

Functional role of a conserved arginine residue located on a mobile loop of alkanesulfonate monooxygenase.

The structure of the flavin-dependent alkanesulfonate monooxygenase (SsuD) exists as a TIM-barrel structure with an insertion region located over the active site that contains a conserved arginine (Arg297) residue present in all SsuD homologues. Substitution of Arg297 with alanine (R297A SsuD) or lysine (R297K SsuD) was performed to determine the functional role of this conserved residue in SsuD catalysis. While the more conservative R297K SsuD possessed a lower k(cat)/K(m) value (0.04 ± 0.01 µM(-1) min(-1)) relative to wild-type (1.17 ± 0.22 µM(-1) min(-1)), there was no activity observed with the R297A SsuD variant. Each of the arginine variants had similar K(d) values for flavin binding as wild-type SsuD (0.32 ± 0.15 µM), but there was no measurable binding of octanesulfonate. The low levels of activity for the R297A and R297K SsuD variants correlated with the absence of any detectable C4a-(peroxy)flavin formation in stopped-flow kinetic studies. Single-turnover experiments were performed in the presence of SsuE to evaluate both the reductive and oxidative half-reaction. With wild-type SsuD a lag phase is observed following the reductive half-reaction by SsuE that represents flavin transfer or conformational changes associated with the binding of substrates. Evaluation of the Arg297 SsuD variants in the presence of SsuE showed no lag phase following reduction by SsuE, and the flavin was oxidized immediately following the reductive half-reaction. These results corresponded with a lack of detectable changes in the proteolytic susceptibility of R297A and R297K SsuD in the presence of reduced flavin and/or octanesulfonate, signifying the absence of a conformational change in these variants with the substitution of Arg297.