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

Autor: Aritsara Jaruwat, Panu Pimviriyakul, Penchit Chitnumsub, Pimchai Chaiyen
Rok vydání: 2021
Předmět:
NpcA
4-nitrophenol monooxygenase from Rhodococcus opacus SAO101
TftD
2
4
5-trichlorophenol 4-monooxygenase from Burkholderia cepacia AC1100
HadA
a flavin monooxygenase component from Raltonia pikettii
FADH–
reduced form of flavin adenine dinucleotide
Biochemistry
biodegradation
HPs
halogenated phenols
TtHpaB
p-hydroxyphenylacetate hydroxylase from Thermus thermophilus HB8
Mixed Function Oxygenases
Hydroxylation
chemistry.chemical_compound
enzyme kinetics
nitrophenol
PheA1
phenol hydroxylase from Bacillus thermoglucosidasius A7
4NP
4-nitrophenol
EcHpaB
p-hydroxyphenylacetate hydroxylase from Escherichia coli
Structural motif
Site-directed mutagenesis
HadX
a flavin reductase component from Raltonia pikettii
ReTcpA
2
4
6-trichlorophenol 4-monooxygenase from Ralstonia eutropha JMP134
quadruple π-stacking
C2
p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii
4CP
4-chlorophenol
Flavin-Adenine Dinucleotide
inhibition mechanism
FAD
oxidized form of flavin adenine dinucleotide
site-directed mutagenesis
4HPA
p-hydroxyphenylacetate
Research Article
halogenated phenol
crystal structure
Stereochemistry
HQ
hydroquinone
Flavin group
Catalysis
NpdA2
p-nitrophenol monooxygenase from Arthrobacter sp. Strain JS443
DcmB1
two-component flavin monooxygenase from Rhodococcus sp. JT-3
Flavins
Enzyme kinetics
Molecular Biology
Glc-6-PD
glucose-6-phosphate dehydrogenase
Dehalogenase
BQ
benzoquinone
Glc-6-P
glucose-6-phosphate
Mutagenesis
Substrate (chemistry)
HnpA
FAD-dependent two-component monooxygenase from Cupriavidus sp. CNP-8
Cell Biology
PcpB
pentachlorophenol hydroxylase from Sphingobium chlorophenolicum
flavin-dependent monooxygenase/dehalogenase
NP
nitrophenol
CphC-I
a two-component flavin monooxygenase from Arthrobacter chlorophenolicus A6
Kinetics
chemistry
CnTcpA
2
4
6-trichlorophenol monooxygenase from C. nantongensis X1T
Biocatalysis
NpsA1
p-nitrophenol monooxygenase from Rhodococcus sp. strain PN1
Zdroj: The Journal of Biological Chemistry
ISSN: 1083-351X
Popis: 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 (HadAWT) co-complexed with reduced FAD (FADH–) and 4-nitrophenol (4NP) (HadAWT−FADH–−4NP) was solved at 2.3-A 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 HadAPhe441Val−FADH–−4NP complex at 2.3-A resolution. Although 4NP can still bind to this variant, the quadruple π-stacking motif was disrupted. All HadAPhe441 variants lack substrate inhibition behavior, confirming that quadruple π-stacking is a main cause of dead-end complex formation. Moreover, the activities of these HadAPhe441 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.
Databáze: OpenAIRE
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