ArticleThe Journal of biological chemistry2011
Stabilization of C4a-hydroperoxyflavin in a two-component flavin-dependent monooxygenase is achieved through interactions at flavin N5 and C4a atoms.
Article in The Journal of biological chemistry, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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29 citing papers in PubMed, 65 citations in OpenAlex.
- Functional analysis of R144 in the flavin oxidation mechanism of a flavin-dependent N-monooxygenase.Protein science : a publication of the Protein Society · 2025Article
- Unlocking the catalytic precision of ligand-controlled enzymatic halogenation.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase.Biochemistry · 2024Article
- 4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for VersatileInternational journal of molecular sciences · 2024Review
- Unifying and versatile features of flavin-dependent monooxygenases: Diverse catalysis by a common C4a-(hydro)peroxyflavin.The Journal of biological chemistry · 2023Review
- Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase fromThe Journal of biological chemistry · 2020Article
- Structural and chemical trapping of flavin-oxide intermediates reveals substrate-directed reaction multiplicity.Protein science : a publication of the Protein Society · 2020Article
- Viscous Media Slow Down the Decay of the Key Intermediate in Bacterial Bioluminescent Reaction.Doklady. Biochemistry and biophysics · 2020Article
- Tuning of pThe Journal of biological chemistry · 2020Article
- An overview of microbial indigo-forming enzymes.Applied microbiology and biotechnology · 2020Review
- The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level.Acta crystallographica. Section D, Structural biology · 2019Article
- Flavin oxidation in flavin-dependent N-monooxygenases.Protein science : a publication of the Protein Society · 2019Article
- Monooxygenation of aromatic compounds by flavin-dependent monooxygenases.Protein science : a publication of the Protein Society · 2019Review
- Two-Component FAD-Dependent Monooxygenases: Current Knowledge and Biotechnological Opportunities.Biology · 2018Review
- Inactivation mechanism of N61S mutant of human FMO3 towards trimethylamine.Scientific reports · 2017Article
- Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophileThe Journal of biological chemistry · 2017Article
- Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA.The Journal of biological chemistry · 2017Article
- Initial investigations of C4a-(hydro)peroxyflavin intermediate formation by dibenzothiophene monooxygenase.Biochemical and biophysical research communications · 2016Article
- Flavin-Dependent Redox Transfers by the Two-Component Diketocamphane Monooxygenases of Camphor-Grown Pseudomonas putida NCIMB 10007.Microorganisms · 2016Article
- Structure of DnmZ, a nitrososynthase in the Streptomyces peucetius anthracycline biosynthetic pathway.Acta crystallographica. Section F, Structural biology communications · 2015Article
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5 authors at 3 institutions in 2 countries.
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Abstract
p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C(2)), His-396 is 4.5 Å from the flavin C4a locus, whereas Ser-171 is 2.9 Å from the flavin N5 locus. We investigated the roles of these two residues in the stability of the C4a-hydroperoxy-FMN intermediate. The results indicated that the rate constant for C4a-hydroperoxy-FMN formation decreased ~30-fold in H396N, 100-fold in H396A, and 300-fold in the H396V mutant, compared with the wild-type enzyme. Lesser effects of the mutations were found for the subsequent step of H(2)O(2) elimination. Studies on pH dependence showed that the rate constant of H(2)O(2) elimination in H396N and H396V increased when pH increased with pK(a) >9.6 and >9.7, respectively, similar to the wild-type enzyme (pK(a) >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H(2)O(2) elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H(2)O(2) elimination in S171A and S171T were ~1400-fold and 8-fold greater than the wild type, respectively. Studies on the pH dependence of S171A with oxygen showed that the rate constant of H(2)O(2) elimination increased with pH rise and exhibited an approximate pK(a) of 8.0. These results indicated that the interaction of the hydroxyl group side chain of Ser-171 and flavin N5 is required for the stabilization of C4a-hydroperoxy-FMN. The double mutant S171A/H396V reacted with oxygen to directly form the oxidized flavin without stabilizing the C4a-hydroperoxy-FMN intermediate, which confirmed the findings based on the single mutation that His-396 was important for formation and Ser-171 for stabilization of the C4a-hydroperoxy-FMN intermediate in C(2).
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