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

Abstract

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.

Radical SAM-dependent ether crosslink in daropeptide biosynthesis

“…The dAdo radical produced from the second SAM molecules then abstracts the hydrogen from the β-hydroxyl group of DarA- 4 to produce an oxygen-centered radical X , which then attacks the C7 indole ring of Trp49 to install the C–O–C linkage (Fig. 4 ), in a way similar to that proposed in glycopeptide biosynthesis 35 , 36 . It is noteworthy that although hydrogen abstraction from an alcohol hydroxyl group is rare in biochemistry, this type of chemistry has been demonstrated in NosL catalysis with unnatural substrates 37 – 39 .…”

Current Progress in the Chemoenzymatic Synthesis of Natural Products

“…Peptide and amino acid-based natural products have been some of the most versatile and important natural products used in the clinical setting including molecules such as Vancomycin and Insulin [ 57 ]. As such, there is a rich library of literature involving their syntheses and specifically their chemoenzymatic syntheses [ 58 ].…”

Strategic Enzymatic Transformations in Modern Natural Product Synthesis

“…For instance, the Gulder group furnished the bityrosine motif of arylomycin by enzymatic coupling following construction of the peptide backbone by solid phase synthesis. 17 The groups of Seyedsayamdost 18 and Tailhades and Cryle 19 independently used OxyB, a P450 from vancomycin biosynthesis, to introduce a variety of different aryl connections into linear peptides with (sometimes only distant) structural similarity to the native substrate. Complementing the previously mentioned work with the oxygenase AsqJ, the Gulder group employed this enzyme for ring contractions yielding quinazolinones through a radical mechanism relying on the absence of stabilizing effects in non-native substrates.…”