Use of a Tyrosine Analogue To Modulate the Two Activities of a Nonheme Iron Enzyme OvoA in Ovothiol Biosynthesis, Cysteine Oxidation versus Oxidative C-S Bond Formation

Abstract

Ovothiol is a histidine thiol derivative. The biosynthesis of ovothiol involves an extremely efficient trans-sulfuration strategy. The nonheme iron enzyme OvoA catalyzed oxidative coupling between cysteine and histidine is one of the key steps. Besides catalyzing the oxidative coupling between cysteine and histidine, OvoA also catalyzes the oxidation of cysteine to cysteine sulfinic acid (cysteine dioxygenase activity). Thus far, very little mechanistic information is available for OvoA-catalysis. In this report, we measured the kinetic isotope effect (KIE) in OvoA-catalysis using the isotopically sensitive branching method. In addition, by replacing an active site tyrosine (Tyr417) with 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propanoic acid (MtTyr) through the amber suppressor mediated unnatural amino acid incorporation method, the two OvoA activities (oxidative coupling between cysteine and histidine, and cysteine dioxygenase activity) can be modulated. These results suggest that the two OvoA activities branch out from a common intermediate and that the active site tyrosine residue plays some key roles in controlling the partitioning between these two pathways.

Figure 1

Use of the isotopically sensitive branching method in OvoA-mechanistic studies. P1 represents compound 7 and P2 represents cysteine sulfinic acid 11 in this study.

Figure 2

Measurement of the KIE using the isotopically sensitive branching method. A. Using [U-²H₅]-histidine as the substrate. On the top are the conditions of the two reactions and at the bottom are the mass spectra of cysteine sulfinic acid 11 and the coupling product 7. B. Solvent KIE measurement by running the reactions in H₂O buffer and D₂O buffer, respectively. Any ion intensity presented here is the average height of twenty mass spectrometry scans.

Figure 3

KIE measurement using OvoA Y417MtTyr variant as the enzyme. A. ¹³C-NMR spectra of the reaction mixture. B. Mass spectra of the coupling product 7 (left panel) and cysteine sulfinic acid 11 (right panel) by running the reactions with unlabeled substrates (histidine and cysteine) or labeled substrates ([U-²H₅]-histidine and [β-¹³C]-cysteine), respectively. C. Mass spectra of the coupling product 7 (left panel) and cysteine sulfinic acid 11 (right panel) by running the reactions in H₂O buffer or D₂O buffer, respectively. Any ion intensity presented here is the average height of twenty mass spectrometry scans.

SCHEME 1

A. Two aerobic ergothioneine biosynthetic pathways in bacteria and fungi; B. The anaerobic ergothioneine biosynthetic pathway; C. The proposed ovothiol biosynthetic pathway. D. MtTyr used in this study and the Cys-Tyr crosslink in cysteine dioxygenase.

Scheme 2

Proposed OvoA mechanistic models.