Abstract
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-hydroxylase (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.
Highlights
Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms
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
The results indicate that the phenolate state of 4F3HB is stabilized by 1.6 pH units upon binding to 3-hydroxybenzoate 6-hydroxylase (3HB6H), consistent with prior work [32]
Summary
Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. Hydroxylation of aromatic compounds is important in catabolism and transformation of xenobiotics, for a wide variety of organisms Such reactions are mostly catalyzed by metal-, pterin-, and flavin-dependent enzymes [1,2,3,4,5]. Members of the first type utilize flavin as a cofactor, which remains bound throughout the catalytic cycle, and among the second type the oxygenase component binds reduced flavin as a substrate and releases oxidized flavin as a product Both types of hydroxylases catalyze the reaction of reduced flavin with O2 to
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