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

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.

Coinducible Catabolism of 1-Naphthol via Synergistic Regulation of the Initial Hydroxylase Genes in Sphingobium sp. Strain B2

“…In addition, bacteria such as Escherichia coli have been reported to produce acidic by-products (e.g., acetate) during glucose metabolism (35). By coincidence, the optimum pH for NdcA1A2 activity was 5.0, which was different from that of most monooxygenases, with an optimal pH range of 6.0 to 8.0 (36,37). Nevertheless, the relationships between ndcA1A2 transcription and primary carbon source metabolism still need further investigation, and the detailed regulatory mechanisms for the precise catabolism of 1-naphthol by the two regulators (NdcR and NdcS) will be elaborated in another study.…”

Three Rings to Rule Them All: How Versatile Flavoenzymes Orchestrate the Structural Diversification of Natural Products

“…It is a tantalizing idea to employ “talented” tailoring enzymes for the production of natural product derivatives via biotechnological approaches in vitro or in vivo , e.g., by broadening the substrate scope. However, there are significant hurdles that impede such endeavors; e.g., group A FPMOs such as GrhO5 or RslO9 feature complex catalytic cycles and typically only react with NAD­(P)H in the presence of their native substrate (see refs − , , and for further information). It is currently unclear how relaxed the substrate specificity for such enzymes is and if these proofreading mechanisms can be bypassed by maintaining certain substrate features.…”

Flavoprotein Monooxygenases and Halogenases

“…Most of these enzymes, spread over both clades, catalyze hydroxylation reactions and are involved in the microbial degradation of (halo)phenols [11,116]. FAD-dependent 4-hydroxyphenylacetate 3-hydroxylase (C2-HpaH; EC 1.14.14.9) is the prototype aromatic hydroxylase of group D. The reaction mechanism and structurefunction relationship of C2-HpaH has been studied in great detail, and its mode of oxygen and substrate activation has been compared with that of single-component flavoprotein aromatic hydroxylases [117,118]. C2-HpaH has also been engineered for biocatalytic applications.…”