Insights into the catalytic mechanism of chlorophenol 4-monooxygenase: a quantum mechanics/molecular mechanics study

Li, Yanwei; Zhang, Ruiming; Du, Likai; Zhang, Qingzhu; Wang, Wenxing

doi:10.1039/c4ra16165c

Cited by 11 publications

(8 citation statements)

References 45 publications

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“…This suggests that, although C4a-hydroperoxy-FAD can be formed in HadA His290Asn , the enzyme mostly eliminates H 2 O 2 via an unproductive pathway without catalyzing dehalogenation/denitration, possibly due to the lack of His290. Therefore, we conclude that His290 is important for stabilization of C4a-hydroperoxy-FAD as well as for facilitating product formation in dehalogenation/denitration reactions as previously proposed ( 8 , 17 , 41 ).…”

Section: Resultssupporting

confidence: 82%

“…Based on apoenzyme structures, His290 in HadA and His289 in TftD were previously proposed as a general base to abstract a proton from a phenolic substrate, which triggers delocalization of a lone pair of electrons from O1 to the C4 position of the substrate, facilitating monooxygenation by C4a-hydroperoxy-FAD (shown in Fig. 1 ) ( 8 , 17 , 41 ). To elucidate the role of His290 in catalysis, we constructed and overexpressed nine His290 variants (listed in Table.…”

Section: Resultsmentioning

confidence: 99%

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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

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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.

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

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

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“…5 and Table 2), the hydroxylation step is a major rate-limiting step of the HadA reaction. This result agrees with computational calculation models of the reaction of TftD in which the hydroxylation step was proposed to be the rate-determining step for dechlorination (50). The involvement of C4a-flavin adduct species in the HadA reaction makes its mechanism unique from the mechanisms of reductive dehalogenation performed by tyrosine deiodinase from human (51, 52) and glutathione-dependent dehalogenase from S. chlorophenolicum (53).…”

Section: Discussionsupporting

confidence: 83%

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Pimviriyakul

Thotsaporn

Sucharitakul

et al. 2017

Journal of Biological Chemistry

108

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Edited by F. Peter GuengerichThe accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.

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“…† The QM/MM results indicate that different snapshots can be associated with different barriers. To analyze the barrier difference, Boltzmann-weighted averaging method on the rate constant was used: [45][46][47][48] …”

Section: Resultsmentioning

confidence: 99%

Insight into the catalytic mechanism of meta-cleavage product hydrolase BphD: a quantum mechanics/molecular mechanics study

Zhang

et al. 2015

RSC Adv.

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The catalytic mechanism of BphD (meta-cleavage product hydrolase, the fourth enzyme of the biphenyl catabolic pathway) toward its natural substrate 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) was investigated in atomistic detail by the QM/MM approach. The calculated Boltzmannweighted average barriers favor a substrate-assisted acylation mechanism, and the most feasible acylation pathway involves a catalytic triad (Ser-His-Asp). The product (2-hydroxypenta-2,4-dienoic acid) of the acylation process is replaced by three water molecules, and one of which is involved in the deacylation process. The established acylation and deacylation mechanism may shed light on investigating the degradation processes of wt BphD toward hundreds of other differently chlorinated HOPDA. The roles of seventeen residues during the catalytic process of wt BphD toward HOPDA were also reported in search of new promising experimental mutation targets for the improvement of BphD catalytic efficiency.

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Insights into the catalytic mechanism of chlorophenol 4-monooxygenase: a quantum mechanics/molecular mechanics study

Cited by 11 publications

References 45 publications

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

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

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Insight into the catalytic mechanism of meta-cleavage product hydrolase BphD: a quantum mechanics/molecular mechanics study

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