Dissecting the low catalytic capability of flavin-dependent halogenases

Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I-, Br-, and Cl- but not F- to form C4a-hydroxyflavin and HOX. Our experiments revealed that I- reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.</p

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 <i>p</i>-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 (∼10⁶ 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 <i>p</i>‑Hydroxyphenylacetate-3-hydroxylase

The protonation status of the peroxide moiety in C4a-(hydro)­peroxyflavin of <i>p</i>-hydroxyphenylacetate-3-hydroxylase can be directly monitored using transient kinetics. The p<i>K</i>_a 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

Tuning of pK_a values activates substrates in flavin-dependent aromatic hydroxylases

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19F NMR in conjunction with fluorinated substrate analogs to directly measure pKa values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hy-droxylase (3HB6H) depresses the pKa of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pKa of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C2 component of HPAH. 19F NMR also revealed a pKa of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions. Our implementation of 19F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.</p

Protonation status and control mechanism of flavin–oxygen intermediates in the reaction of bacterial luciferase

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167808/1/febs15653.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167808/2/febs15653_am.pd

<i>p</i>‑Hydroxyphenylacetate 3‑Hydroxylase as a Biocatalyst for the Synthesis of Trihydroxyphenolic Acids

Trihydroxyphenolic acids such as 3,4,5-trihydroxycinnamic acid (3,4,5-THCA) <b>4c</b> and 2-(3,4,5-trihydroxyphenyl)­acetic acid (3,4,5-THPA) <b>2c</b> are strong antioxidants that are potentially useful as medicinal agents. Our results show that <i>p</i>-hydroxyphenylacetate (HPA) 3-hydroxylase (HPAH) from <i>Acinetobacter baumannii</i> can catalyze the syntheses of 3,4,5-THPA <b>2c</b> and 3,4,5-THCA <b>4c</b> from 4-HPA <b>2a</b> and <i>p</i>-coumaric acid <b>4a</b>, respectively. The wild-type HPAH can convert 4-HPA <b>2a</b> completely into 3,4,5-THPA <b>2c</b> within 100 min (total turnover number (TTN) of 100). However, the wild-type enzyme cannot efficiently synthesize 3,4,5-THCA <b>4c</b>. To improve the efficiency, the oxygenase component of HPAH (C₂) was rationally engineered in order to maximize the conversion of <i>p</i>-coumaric acid <b>4a</b> to 3,4,5-THCA <b>4c</b>. Results from site-directed mutagenesis studies showed that Y398S is significantly more effective than the wild-type enzyme for the synthesis of 3,4,5-THCA <b>4c</b>; it can catalyze the complete bioconversion of <i>p</i>-coumaric acid <b>4a</b> to 3,4,5-THCA <b>4c</b> within 180 min (TTN ∼ 23 at 180 min). The yield and stability of 3,4,5-THPA <b>2c</b> and 3,4,5-THCA <b>4c</b> were significantly improved in the presence of ascorbic acid. Thermostability studies showed that the wild-type C₂ was very stable and remained active after incubation at 30, 35, and 40 °C for 24 h. Y398S was moderately stable because its activity was retained for 24 h at 30 °C and for 15 h at 35 °C. Transient kinetic studies using stopped-flow spectrophotometry indicated that the key improvement in the reaction of Y398S with <i>p</i>-coumaric acid <b>4a</b> lies within the protein–ligand interaction. Y398S binds to <i>p</i>-coumaric acid <b>4a</b> with higher affinity than the wild-type enzyme, resulting in a shift in equilibrium toward favoring the productive coupling path instead of the path leading to wasteful flavin oxidation