A Single-Site Mutation at Ser146 Expands the Reactivity of the Oxygenase Component of <i>p</i>-Hydroxyphenylacetate 3-Hydroxylase

Dhammaraj, Taweesak; Pinthong, Chatchadaporn; Visitsatthawong, Surawit; Tongsook, Chanakan; Surawatanawong, Panida; Chaiyen, Pimchai

doi:10.1021/acschembio.6b00402

Cited by 27 publications

(29 citation statements)

References 31 publications

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“…In another two-component flavin-dependent monooxygenase, p -hydroxyphenylacetate hydroxylase (C 2 ), His 120 was identified as a residue which potentially serves as an active site base to abstract a proton from the phenolic moiety. 85 , 86 Overall, the key catalytic features to deprotonate the phenolic substrate to facilitate the hydroxylation reaction in two-component and single-component monooxygenases are completely different.…”

Section: Discussionmentioning

confidence: 99%

Oxidative dehalogenation and denitration by a flavin-dependent monooxygenase is controlled by substrate deprotonation

2018

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Section: Discussionmentioning

confidence: 99%

Oxidative dehalogenation and denitration by a flavin-dependent monooxygenase is controlled by substrate deprotonation

2018

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“…Furthermore, the individual rate constants associated with each step and the mechanism of reduced flavin transfer between the two components have been reported (24 -30). This knowledge has been useful for applying HPAH for the synthesis of bioactive phenolic acids and in the hydroxylation of aniline derivatives (31,32).…”

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confidence: 99%

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Pimviriyakul

Thotsaporn

Sucharitakul

et al. 2017

Journal of Biological Chemistry

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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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“…For example, cyclohexanone monooxygenase (and other Baeyer-Villiger flavoprotein catalysts) requires a deprotonated C4a-peroxyflavin (C4a-OO − ) intermediate that is favored at high pH, rather than a hydroperoxyflavin (C4a-OOH),42 and in the flavin-containing monooxygenase enzyme SidA, the C4a-OOH hydroxylates the substrate more efficiently at a higher pH 43. In contrast, the hydroxylation efficiency of a modified oxygenase component of p -hydroxyphenylacetate-3-hydroxylase is more efficient at a lower pH 44. To examine whether any steps in the BluB reaction are influenced by pH, we conducted stopped-flow analysis on reactions initiated by mixing FMNH 2 -bound BluB using buffers ranging from 6.0 to 9.5.…”

Section: Resultsmentioning

confidence: 99%

Unique Biochemical and Sequence Features Enable BluB To Destroy Flavin and Distinguish BluB from the Flavin Monooxygenase Superfamily

2018

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Vitamin B (cobalamin) is an essential micronutrient for humans that is synthesized by only a subset of bacteria and archaea. The aerobic biosynthesis of 5,6-dimethylbenzimidazole, the lower axial ligand of cobalamin, is catalyzed by the "flavin destructase" enzyme BluB, which fragments reduced flavin mononucleotide following its reaction with oxygen to yield this ligand. BluB is similar in sequence and structure to members of the flavin oxidoreductase superfamily, yet the flavin destruction process has remained elusive. Using stopped-flow spectrophotometry, we find that the flavin destructase reaction of BluB from Sinorhizobium meliloti is initiated with canonical flavin-O chemistry. A C4a-peroxyflavin intermediate is rapidly formed in BluB upon reaction with O, and has properties similar to those of flavin-dependent hydroxylases. Analysis of reaction mixtures containing flavin analogues indicates that both formation of the C4a-peroxyflavin and the subsequent destruction of the flavin to form 5,6-dimethylbenzimidazole are influenced by the electronic properties of the flavin isoalloxazine ring. The flavin destruction phase of the reaction, which results from the decay of the C4a-peroxyflavin intermediate, occurs more efficiently at pH >7.5. Furthermore, the BluB mutants D32N and S167G are specifically impaired in the flavin destruction phase of the reaction; nevertheless, both form the C4a-peroxyflavin nearly quantitatively. Coupled with a phylogenetic analysis of BluB and related flavin-dependent enzymes, these results demonstrate that the BluB flavin destructase family can be identified by the presence of active site residues D32 and S167.

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A Single-Site Mutation at Ser146 Expands the Reactivity of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Cited by 27 publications

References 31 publications

Oxidative dehalogenation and denitration by a flavin-dependent monooxygenase is controlled by substrate deprotonation

Oxidative dehalogenation and denitration by a flavin-dependent monooxygenase is controlled by substrate deprotonation

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Unique Biochemical and Sequence Features Enable BluB To Destroy Flavin and Distinguish BluB from the Flavin Monooxygenase Superfamily

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