Tuning of pKa values activates substrates in flavin-dependent aromatic hydroxylases

Warintra Pitsawong, Pirom Chenprakhon, Taweesak Dhammaraj, Dheeradhach Medhanavyn, Jeerus Sucharitakul, Chanakan Tongsook, Willem J.H. van Berkel, Pimchai Chaiyen, Anne Frances Miller, Ruma Banerjee

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19F NMR in conjunction with fluorinated substrate analogs to directly measure pKa values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hy-droxylase (3HB6H) depresses the pKa of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pKa of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C2 component of HPAH. 19F NMR also revealed a pKa of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions. Our implementation of 19F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.

Original languageEnglish
Pages (from-to)3965-3981
Number of pages17
JournalJournal of Biological Chemistry
Issue number12
StatePublished - Mar 20 2020

Bibliographical note

Publisher Copyright:
© 2020 Pitsawong et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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