Trihydroxyphenolic acids such as 3,4,5-trihydroxycinnamic acid (3,4,5-THCA) 4c and 2-(3,4,5-trihydroxyphenyl)acetic acid (3,4,5-THPA) 2c are strong antioxidants that are potentially useful as medicinal agents. Our results show that p-hydroxyphenylacetate (HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii can catalyze the syntheses of 3,4,5-THPA 2c and 3,4,5-THCA 4c from 4-HPA 2a and p-coumaric acid 4a, respectively. The wildtype HPAH can convert 4-HPA 2a completely into 3,4,5-THPA 2c within 100 min (total turnover number (TTN) of 100). However, the wild-type enzyme cannot efficiently synthesize 3,4,5-THCA 4c. To improve the efficiency, the oxygenase component of HPAH (C 2 ) was rationally engineered in order to maximize the conversion of p-coumaric acid 4a to 3,4,5-THCA 4c. Results from site-directed mutagenesis studies showed that Y398S is significantly more effective than the wildtype enzyme for the synthesis of 3,4,5-THCA 4c; it can catalyze the complete bioconversion of p-coumaric acid 4a to 3,4,5-THCA 4c within 180 min (TTN ∼ 23 at 180 min). The yield and stability of 3,4,5-THPA 2c and 3,4,5-THCA 4c were significantly improved in the presence of ascorbic acid. Thermostability studies showed that the wild-type C 2 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 p-coumaric acid 4a lies within the protein−ligand interaction. Y398S binds to pcoumaric acid 4a 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.

The oxygenase component (C) of p-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of various phenolic acids. In this report, we found that substitution of a residue close to the phenolic group binding site to yield the S146A variant resulted in an enzyme that is more effective than the wild-type in catalyzing the hydroxylation of 4-aminophenylacetate (4-APA). Product yields for both wild-type and S146A enzymes are better at lower pH values. Multiple turnover reactions of the wild-type and S146A enzymes indicate that both enzymes first hydroxylate 3-APA to give 3-hydroxy-4-aminophenylacetate (3-OH-4-APA), which is further hydroxylated to give 3,5-dihydroxy-4-aminophenylacetate, similar to the reaction of C with 4-HPA. Stopped-flow experiments showed that 4-APA can only bind to the wild-type enzyme at pH 6.0 and not at pH 9.0, while it can bind to S146A under both pH conditions. Rapid-quench flow results indicate that the wild-type enzyme has low reactivity toward 4-APA hydroxylation, with a hydroxylation rate constant (k) for 4-APA of 0.028 s compared to 17 s for 4-HPA, the native substrate. In contrast, for S146A, the hydroxylation rate constants for both substrates are very similar (2.6 s for 4-HPA versus 2.5 s for 4-APA). These data indicate that Ser146 is a key catalytic residue involved in optimizing C reactivity toward a phenolic compound. Removing this hydroxyl group expands C activity toward a non-natural aniline substrate. This understanding should be helpful for future rational engineering of other two-component flavin-dependent monooxygenases that have this conserved Ser residue.

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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-hydroxylase (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.

-Hydroxyphenylacetate 3-hydroxylase component 1 () is a useful enzyme for generating reduced flavin and NAD intermediates. In this study, poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) were used to encapsulate the (PLGA- NPs). Enzymatic activity, stability, and reusability of PLGA- NPs prepared using three different methods [oil in water (o/w), water in oil in water (w/o/w), and solid in oil in water (s/o/w)] were compared. The s/o/w provided the optimal conditions for encapsulation of (PLGA- NPs), giving the highest enzyme activity, stability, and reusability. The s/o/w method improves enzyme activity ∼11 and 9-fold compared to w/o/w (PLGA- NPs) and o/w (PLGA- NPs). In addition, s/o/w prepared PLGA- NPs could be reused 14 times with nearly 50% activity remaining, a much higher reusability compared to PLGA- NPs and PLGA- NPs. These nanovesicles were successfully utilised to generate reduced flavin mononucleotide (FMN) and supply this cofactor to a hydroxylase enzyme that has application for synthesising anti-inflammatory compounds. Therefore, this recycling biocatalyst prepared using the s/o/w method is effective and has the potential for use in combination with other enzymes that require reduced FMN. Application of PLGA- NPs may be possible in additional biocatalytic processes for chemical or biochemical production.

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