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

Chenprakhon, Pirom; Dhammaraj, Taweesak; Chantiwas, Rattikan; Chaiyen, Pimchai

doi:10.1016/j.abb.2017.03.004

Cited by 11 publications

(9 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, engineering the two-component enzyme p-hydroxyphenylacetate-3-hydroxylase from Acinetobacter baumannii by rational-design single-point mutation could expand the substrate scope for value-added compound synthesis. 32,33 The results reported herein elucidate the function of Arg208, which is proposed to be involved in substrate binding. HadA Arg208 variants were successfully constructed by site-directed mutagenesis.…”

Section: Hydroxylation Rate In Hada Arg208 Mutants With 4npmentioning

confidence: 63%

“…The in‐depth investigation of specific functions of amino acid residues in HadA could provide universal information for enzymes in this family, which could be developed in various applications. For example, engineering the two‐component enzyme p ‐hydroxyphenylacetate‐3‐hydroxylase from Acinetobacter baumannii by rational‐design single‐point mutation could expand the substrate scope for value‐added compound synthesis 32,33 …”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Role of conserved arginine in HadA monooxygenase for 4‐nitrophenol and 4‐chlorophenol detoxification

et al. 2022

View full text Add to dashboard Cite

HadA monooxygenase is involved in the initial step of the biodegradation pathway of toxic nitrophenols and halogenated phenols. HadA catalyzes the O2‐dependent denitration of nitrophenols and dehalogenation of halogenated phenols via the hydroquinone pathway. Based on bioinformatics and structural analysis, Arg208 of HadA is located at the proper position for substrate stabilization. This arginine is conserved among hydroquinone pathway‐specific enzymes for toxicant detoxification. In this study, the function of Arg208 in HadA was determined by a single‐point mutation creating HadAArg208 variants. 4‐Nitrophenol was mineralized by HadAArg208 variants that contain side chains as a positive charge and hydrogen‐bond donor, whereas 4‐chlorophenol strictly required a positively charged environment for detoxification. Transient kinetic results indicated that the biodetoxification ability of HadAArg208 variants was diminished due to the slowing down of denitration/dehalogenation. The substrate‐binding mode and affinity energy were evaluated by molecular docking. The findings were consistent with the experimental results indicating that arginine is the most fit for both 4‐nitrophenol and 4‐chlorophenol binding, whereas the active mutants provide a weaker interaction correlated with their denitration/dehalogenation activities. Altogether, Arg208 plays a role in providing proper chemical interactions to the substrate for binding at an appropriate orientation in the active site of hydroquinone pathway‐specific enzymes. In addition, it is proposed to stabilize nitro groups and halide ions that are released in denitration/dehalogenation reactions. This conserved arginine might be the essential feature for related biocatalysts, which could be fundamental knowledge regarding this enzyme family.

show abstract

Section: Hydroxylation Rate In Hada Arg208 Mutants With 4npmentioning

confidence: 63%

Section: Discussionmentioning

confidence: 99%

Role of conserved arginine in HadA monooxygenase for 4‐nitrophenol and 4‐chlorophenol detoxification

et al. 2022

View full text Add to dashboard Cite

show abstract

“…In the past, engineering of C 2 resulted in variants useful for biocatalysis. The variant Tyr398Ser of C 2 catalyzes formation of trihydroxyphenolic acids with higher efficiency than the wild-type enzyme ( 55 ), whereas single mutations at either Ser146 or Arg263 resulted in C 2 variants, which can use p -aminophenylacetate and p -hydroxyphenylethylamine better than the native substrate 4HPA, respectively ( 56 , 57 ). Mutation of group C flavin-dependent luciferases can also change their properties, such as alteration of emission color or prolonging light intensity of the product, which is useful for biodetection applications ( 58 ).…”

Section: Discussionmentioning

confidence: 99%

Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking

Pimviriyakul

Jaruwat

Chitnumsub

et al. 2021

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

HadA is a flavin-dependent monooxygenase catalyzing hydroxylation plus dehalogenation/denitration, which is useful for biodetoxification and biodetection. In this study, the X-ray structure of wild-type HadA (HadA WT ) co-complexed with reduced FAD (FADH – ) and 4-nitrophenol (4NP) (HadA WT −FADH – −4NP) was solved at 2.3-Å resolution, providing the first full package (with flavin and substrate bound) structure of a monooxygenase of this type. Residues Arg101, Gln158, Arg161, Thr193, Asp254, Arg233, and Arg439 constitute a flavin-binding pocket, whereas the 4NP-binding pocket contains the aromatic side chain of Phe206, which provides π-π stacking and also is a part of the hydrophobic pocket formed by Phe155, Phe286, Thr449, and Leu457. Based on site-directed mutagenesis and stopped-flow experiments, Thr193, Asp254, and His290 are important for C4a-hydroperoxyflavin formation with His290, also serving as a catalytic base for hydroxylation. We also identified a novel structural motif of quadruple π-stacking (π-π-π-π) provided by two 4NP and two Phe441 from two subunits. This motif promotes 4NP binding in a nonproductive dead-end complex, which prevents C4a-hydroperoxy-FAD formation when HadA is premixed with aromatic substrates. We also solved the structure of the HadA Phe441Val −FADH – −4NP complex at 2.3-Å resolution. Although 4NP can still bind to this variant, the quadruple π-stacking motif was disrupted. All HadA Phe441 variants lack substrate inhibition behavior, confirming that quadruple π-stacking is a main cause of dead-end complex formation. Moreover, the activities of these HadA Phe441 variants were improved by ⁓20%, suggesting that insights gained from the flavin-dependent monooxygenases illustrated here should be useful for future improvement of HadA’s biocatalytic applications.

show abstract

“…On this basis, AbHpaB was rationally designed to obtain the Y398S mutant, with which the conversion yield of p -coumarate to 3,4,5-trihydroxy-cinamate was increased from 26 to 50% . In another study, R263E and R263D/Y398D mutants were created to catalyze the ortho-hydroxylation of tyramine and octopamine, respectively . The structure of the FAD-dependent HpaB from Thermus thermophilus HB8 was also solved, and the catalytic mechanism was elucidated .…”

Section: Introductionmentioning

confidence: 99%

“…9 In another study, R263E and R263D/Y398D mutants were created to catalyze the ortho-hydroxylation of tyramine and octopamine, respectively. 10 The structure of the FAD-dependent HpaB from Thermus thermophilus HB8 was also solved, and the catalytic mechanism was elucidated. 11 Specifically, R100-Y104-H142 is involved in substrate stabilization and proton transfer and is highly conserved in homologous sequences.…”

Section: Introductionmentioning

confidence: 99%

Engineering a Prokaryotic Non-P450 Hydroxylase for 3′-Hydroxylation of Flavonoids

Wang

et al. 2022

ACS Synth. Biol.

View full text Add to dashboard Cite

Plant-derived cytochrome P450-dependent flavonoid 3′-hydroxylases are the rate-limiting enzymes in flavonoid biosynthesis. In this study, the large component (HpaB) of a prokaryotic 4-hydroxyphenylacetate (4-HPA) 3-hydroxylase was engineered for efficient 3′-hydroxylation of naringenin. First, we selected four HpaBs through database search and phylogenetic analysis and compared their catalytic activities toward 4-HPA and naringenin. HpaB from Rhodococcus opacus B-4 (RoHpaB) showed better preference toward naringenin. To elucidate the underlying mechanism, we analyzed the structural differences of HpaBs through homologous modeling, molecular docking, and molecular dynamics simulation, and the substrate preference of RoHpaB was mainly attributed to the shorter chain length of loop 212−222 and the larger substrate binding pocket. RoHpaB was further engineered by alanine scanning and structural replacement, and the RoHpaB Y215A variant was obtained, whose catalytic efficiency (k cat /K m ) toward naringenin is 25.3 times higher than that of RoHpaB. In addition, RoHpaB Y215A also showed significantly improved activity toward flavonoids apigenin and kaempferol. This work opens the possibility of using engineered HpaB as a versatile hydroxylase to produce functionalized flavonoids.

show abstract

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

Cited by 11 publications

References 48 publications

Role of conserved arginine in HadA monooxygenase for 4‐nitrophenol and 4‐chlorophenol detoxification

Role of conserved arginine in HadA monooxygenase for 4‐nitrophenol and 4‐chlorophenol detoxification

Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking

Engineering a Prokaryotic Non-P450 Hydroxylase for 3′-Hydroxylation of Flavonoids

Contact Info

Product

Resources

About