Tuning of p
| Autor: | Warintra, Pitsawong, Pirom, Chenprakhon, Taweesak, Dhammaraj, Dheeradhach, Medhanavyn, Jeerus, Sucharitakul, Chanakan, Tongsook, Willem J H, van Berkel, Pimchai, Chaiyen, Anne-Frances, Miller |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Binding Sites
Hydrogen Bonding Hydrogen-Ion Concentration 4-Hydroxybenzoate-3-Monooxygenase Mixed Function Oxygenases Substrate Specificity Kinetics Bacterial Proteins Catalytic Domain Flavins Biocatalysis Mutagenesis Site-Directed Enzymology Rhodococcus Nuclear Magnetic Resonance Biomolecular Phenylacetates |
| Zdroj: | J Biol Chem |
| ISSN: | 1083-351X |
| Popis: | 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 (19)F NMR in conjunction with fluorinated substrate analogs to directly measure pK(a) values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the pK(a) of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. (19)F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pK(a) of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C(2) component of HPAH. (19)F NMR also revealed a pK(a) 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-C(2), respectively, are consistent with pK(a) tuning by one or more H-bonding interactions. Our implementation of (19)F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|