Pirom Chenprakhon

Multiple pathways guide oxygen diffusion into flavoenzyme active sites

Dioxygen (O2) and other gas molecules have a fundamental role in a variety of enzymatic reactions. However, it is only poorly understood which O2 uptake mechanism enzymes employ to promote efficient catalysis and how general this is. We investigated O2 diffusion pathways into monooxygenase and oxidase flavoenzymes, using an integrated computational and experimental approach. Enhanced-statistics molecular dynamics simulations reveal spontaneous protein-guided O2 diffusion from the bulk solvent to preorganized protein cavities. The predicted protein-guided diffusion paths and the importance of key cavity residues for oxygen diffusion were verified by combining site-directed mutagenesis, rapid kinetics experiments, and high-resolution X-ray structures. This study indicates that monooxygenase and oxidase flavoenzymes employ multiple funnel-shaped diffusion pathways to absorb O2 from the solvent and direct it to the reacting C4a atom of the flavin cofactor. The difference in O2 reactivity among dehydrogenases, monooxygenases, and oxidases ultimately resides in the fine modulation of the local environment embedding the reactive locus of the flavin.

Stabilization of C4a-Hydroperoxyflavin in a Two-component Flavin-dependent Monooxygenase Is Achieved through Interactions at Flavin N5 and C4a Atoms

p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C2), His-396 is 4.5 Å from the flavin C4a locus, whereas Ser-171 is 2.9 Å from the flavin N5 locus. We investigated the roles of these two residues in the stability of the C4a-hydroperoxy-FMN intermediate. The results indicated that the rate constant for C4a-hydroperoxy-FMN formation decreased ∼30-fold in H396N, 100-fold in H396A, and 300-fold in the H396V mutant, compared with the wild-type enzyme. Lesser effects of the mutations were found for the subsequent step of H2O2 elimination. Studies on pH dependence showed that the rate constant of H2O2 elimination in H396N and H396V increased when pH increased with pKa >9.6 and >9.7, respectively, similar to the wild-type enzyme (pKa >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H2O2 elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H2O2 elimination in S171A and S171T were ∼1400-fold and 8-fold greater than the wild type, respectively. Studies on the pH dependence of S171A with oxygen showed that the rate constant of H2O2 elimination increased with pH rise and exhibited an approximate pKa of 8.0. These results indicated that the interaction of the hydroxyl group side chain of Ser-171 and flavin N5 is required for the stabilization of C4a-hydroperoxy-FMN. The double mutant S171A/H396V reacted with oxygen to directly form the oxidized flavin without stabilizing the C4a-hydroperoxy-FMN intermediate, which confirmed the findings based on the single mutation that His-396 was important for formation and Ser-171 for stabilization of the C4a-hydroperoxy-FMN intermediate in C2.

Mechanism of Oxygen Activation in a Flavin-Dependent Monooxygenase: A Nearly Barrierless Formation of C4a-Hydroperoxyflavin via Proton-Coupled Electron Transfer

Understanding how flavin-dependent enzymes activate oxygen for their oxidation and oxygenation reactions is one of the most challenging issues in flavoenzymology. Density functional calculations and transient kinetics were performed to investigate the mechanism of oxygen activation in the oxygenase component (C2) of p-hydroxyphenylacetate 3-hydroxylase (HPAH). We found that the protonation of dioxygen by His396 via a proton-coupled electron transfer mechanism is the key step in the formation of the triplet diradical complex of flavin semiquinone and (•)OOH. This complex undergoes intersystem crossing to form the open-shell singlet diradical complex before it forms the closed-shell singlet C4a-hydroperoxyflavin intermediate (C4aOOH). Notably, density functional calculations indicated that the formation of C4aOOH is nearly barrierless, possibly facilitated by the active site arrangement in which His396 positions the proximal oxygen of the (•)OOH in an optimum position to directly attack the C4a atom of the isoalloxazine ring. The nearly barrierless formation of C4aOOH agrees well with the experimental results; based on transient kinetics and Eyring plot analyses, the enthalpy of activation for the formation of C4aOOH is only 1.4 kcal/mol and the formation of C4aOOH by C2 is fast (∼10(6) M(-1) s(-1) at 4 °C). The calculations identified Ser171 as the key residue that stabilizes C4aOOH by accepting a hydrogen bond from the H(N5) of the isoalloxazine ring. Both Ser171 and Trp112 facilitate H2O2 elimination by donating hydrogen bonds to the proximal oxygen of the OOH moiety during the proton transfer. According to our combined theoretical and experimental studies, the existence of a positively charged general acid at the position optimized for facilitating the proton-coupled electron transfer has emerged as an important catalytic feature for the oxygen activation process in flavin-dependent enzymes.

Control of C4a-hydroperoxyflavin protonation in the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase.

The protonation status of the peroxide moiety in C4a-(hydro)peroxyflavin of p-hydroxyphenylacetate-3-hydroxylase can be directly monitored using transient kinetics. The pKa for the wild-type (WT) enzyme is 9.8 ± 0.2, while the values for the H396N, H396V, and H396A variants are 9.3 ± 0.1, 7.3 ± 0.2, and 7.1 ± 0.2, respectively. The hydroxylation efficiency of these mutants is lower than that of the WT enzyme. Solvent kinetic isotope effect studies indicate that proton transfer is not the rate-limiting step in the formation of C4a-OOH. All data suggest that His396 may act as an instantaneous proton provider for the proton-coupled electron transfer that occurs before the transition state of C4a-OOH formation.

Biotransformation of Plant-Derived Phenolic Acids

Phenolic acids are abundant biomass feedstock that can be derived from the processing of lignin or other byproducts from agro‐industrial waste. Although phenolic acids such as p‐hydroxybenzoic acid, p‐coumaric acid, caffeic acid, vanillic acid, cinnamic acid, gallic acid, syringic acid, and ferulic acid can be used directly in various applications, their value can be significantly increased when they are further modified to high value‐added compounds. This review summarizes and discusses the new advances in cell‐free and whole‐cell biocatalysis technologies for reactions important for conversion of phenolic acids including esterification, decarboxylation, amination, halogenation, hydroxylation, and ring‐breakage reactions. The products of these reactions are useful for the pharmaceutical, cosmetic, food, fragrance, and polymer industries. Production of phenolic acids is sustainable, and these processes for their biotransformation are clean technologies that do not produce toxic waste and use less energy than conventional physical and chemical methods. Thus, biotransformation of phenolic acids provides an economically viable and sustainable means for producing useful materials for society.

Hydroxylation of 4-hydroxyphenylethylamine derivatives by R263 variants of the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase

p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to yield 3,4-dihydroxyphenylacetate (DHPA). In this study, we investigated whether variants of the oxygenase component (C2) could catalyze hydroxylation of 4-hydroxyphenylethylamines to synthesize catecholamine derivatives. Single turnover product analysis showed that the R263D variant can catalyze hydroxylation of tyramine to form dopamine with the highest yield (57%). The enzyme was also found to have dual substrate charge specificity because it can also maintain reasonable hydroxylation efficiency of HPA (86%). This property is different from the R263E variant, which can hydroxylate HPA (73%) but not tyramine. The R263A variant can hydroxylate HPA (72%) and tyramine to a small extent (7%). Stopped-flow experiments indicated that tyramine and HPA prefer binding to R263D after C4a-hydroperoxy-FMN formation, while tyramine cannot bind to the wild-type or R263E enzymes. Data also indicate that the hydroxylation rate constant is the rate-limiting step. The R263D variant was used as a starting enzyme for further mutation to obtain other variants for the synthesis of additional catecholamine drugs. The R263D/Y398D double mutant enzyme showed interesting results in that it was able to catalyze the hydroxylation of octopamine to form norepinephrine. However, the enzyme still lacked stereo-selectivity in its reaction.

Monooxygenation of Aromatic Compounds by Flavin-Dependent Monooxygenases: Mechanisms of Flavin-dependent Aromatic Hydroxylases

Many flavoenzymes catalyze hydroxylation of aromatic compounds especially phenolic compounds have been isolated and characterized. These enzymes can be classified as either single‐component or two‐component flavin‐dependent hydroxylases (monooxygenases). The hydroxylation reactions catalyzed by the enzymes in this group are useful for modifying the biological properties of phenolic compounds. This review aims to provide an in‐depth discussion of the current mechanistic understanding of representative flavin‐dependent monooxygenases including 3‐hydroxy‐benzoate 4‐hydroxylase (PHBH, a single‐component hydroxylase), 3‐hydroxyphenylacetate 4‐hydroxylase (HPAH, a two‐component hydroxylase), and other monooxygenases which catalyze reactions in addition to hydroxylation, including 2‐methyl‐3‐hydroxypyridine‐5‐carboxylate oxygenase (MHPCO, a single‐component enzyme that catalyzes aromatic‐ring cleavage), and HadA monooxygenase (a two‐component enzyme that catalyzes additional group elimination reaction). These enzymes have different unique structural features which dictate their reactivity toward various substrates and influence their ability to stabilize flavin intermediates such as C4a‐hydroperoxyflavin. Understanding the key catalytic residues and the active site environments important for governing enzyme reactivity will undoubtedly facilitate future work in enzyme engineering or enzyme redesign for the development of biocatalytic methods for the synthesis of valuable compounds.