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 cross-links. The antibiotic of-last-resort, vancomycin, contains three such cross-links, 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 cross-links share early steps in their catalytic cycles.

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.

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

Tyramine ß-monooxygenase (TßM) 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 TßM, this event is too fast to be rate limiting. However, we have recently shown [Osborne, R. L.; et al. Biochemistry 2013, 52, 1179] that the Tyr216Ala variant of TßM 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 kcat is found in a region where kcat 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 (Ea = 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.

Mapping and Exploiting the Promiscuity of OxyB toward the Biocatalytic Production of Vancomycin Aglycone Variants.

Vancomycin is one of the most important clinical antibiotics in the fight against infectious disease. Its biological activity relies on three aromatic cross-links, which create a cup-shaped topology and allow tight binding to nascent peptidoglycan chains. The cytochrome P450 enzymes OxyB, OxyA, and OxyC have been shown to introduce these synthetically challenging aromatic linkages. The ability to utilize the P450 enzymes in a chemo-enzymatic scheme to generate vancomycin derivatives is appealing but requires a thorough understanding of their reactivities and mechanisms. Herein, we systematically explore the scope of OxyB biocatalysis and report installation of diverse diaryl ether and biaryl cross-links with varying macrocycle sizes and compositions, when the enzyme is presented with modified vancomycin precursor peptides. The structures of the resulting products were determined using one-dimensional/two-dimensional nuclear magnetic resonance spectroscopy, high-resolution mass spectrometry (HR-MS), tandem HR-MS, and isotopic labeling, as well as ultraviolet-visible light absorption and fluorescence emission spectroscopies. An exploration of the biological activities of these alternative OxyB products surprisingly revealed antifungal properties. Taking advantage of the promiscuity of OxyB, we chemo-enzymatically generated a vancomycin aglycone variant containing an expanded macrocycle. Mechanistic implications for OxyB and future directions for creating vancomycin analogue libraries are discussed.