Detection of a Kinetically Competent Compound-I Intermediate in the Vancomycin Biosynthetic Enzyme OxyB

Cytochrome P450 enzymes are abundantly encoded in microbial genomes. Their reactions have two general outcomes, one involving oxygen insertion via a canonical ‘oxygen rebound’ mechanism and a second that diverts from this pathway and leads to a wide array of products, notably intramolecular oxidative crosslinks. The antibiotic of-last-resort vancomycin contains three such crosslinks, which are crucial for biological activity and are installed by the P450 enzymes OxyB, OxyA, and OxyC. The mechanisms of these enzymes have remained elusive in part because of the difficulty in spectroscopically capturing transient intermediates. Using stopped-flow UV/Visible absorption and rapid freeze-quench electron paramagnetic resonance spectroscopies, we show that OxyB generates the highly reactive compound-I intermediate, which can react with a model vancomycin peptide substrate in a kinetically competent fashion to generate product. Our results have implications for the mechanism of OxyB and are in line with the notion that oxygen rebound and oxidative crosslinks share early steps in their catalytic cycles

Solvent and Temperature Probes of the Long-Range Electron-Transfer Step in Tyramine β‑Monooxygenase: Demonstration of a Long-Range Proton-Coupled Electron-Transfer Mechanism

Tyramine β-monooxygenase (TβM) belongs to a family of physiologically important dinuclear copper monooxygenases that function with a solvent-exposed active site. To accomplish each enzymatic turnover, an electron transfer (ET) must occur between two solvent-separated copper centers. In wild-type TβM, this event is too fast to be rate limiting. However, we have recently shown [Osborne, R. L.; et al. <i>Biochemistry</i> <b>2013</b>, <i>52</i>, 1179] that the Tyr216Ala variant of TβM leads to rate-limiting ET. In this study, we present a pH–rate profile study of Tyr216Ala, together with deuterium oxide solvent kinetic isotope effects (KIEs). A solvent KIE of 2 on <i>k</i>_cat is found in a region where <i>k</i>_cat is pH/pD independent. As a control, the variant Tyr216Trp, for which ET is not rate determining, displays a solvent KIE of unity. We conclude, therefore, that the observed solvent KIE arises from the rate-limiting ET step in the Tyr216Ala variant, and show how small solvent KIEs (ca. 2) can be fully accommodated from equilibrium effects within the Marcus equation. To gain insight into the role of the enzyme in the long-range ET step, a temperature dependence study was also pursued. The small enthalpic barrier of ET (<i>E</i>_a = 3.6 kcal/mol) implicates a significant entropic barrier, which is attributed to the requirement for extensive rearrangement of the inter-copper environment during PCET catalyzed by the Tyr216Ala variant. The data lead to the proposal of a distinct inter-domain pathway for PCET in the dinuclear copper monooxygenases

Modulating OxyB-Catalyzed Cross-Coupling Reactions in Vancomycin Biosynthesis by Incorporation of Diverse d‑Tyr Analogues

We report a general method for synthesizing diverse d-Tyr analogues, one of the constituents of the antibiotic vancomycin, using a Negishi cross-coupling protocol. Several analogues were incorporated into the vancomycin substrate–peptide and reacted with the biosynthetic enzymes OxyB and OxyA, which install the characteristic aromatic cross-links. We find that even small structural perturbations are not accepted by OxyA. The same modifications, however, enhance the catalytic capabilities of OxyB leading to the formation of a new macrocycle within the vancomycin framework