The Structure of the Bifunctional Everninomicin Biosynthetic Enzyme EvdMO1 Suggests Independent Activity of the Fused Methyltransferase-Oxidase Domains

Abstract

The orthosomycin family of natural products are decorated polysaccharides with potent antibiotic activity and complex biosynthetic pathways. The defining feature of the orthosomycins is an orthoester linkage between carbohydrate moieties that is necessary for antibiotic activity and is likely formed by a family of conserved oxygenases. Everninomicins are octasaccharide orthosomycins produced by Micromonospora carbonacea that have two orthoester linkages and a methylenedioxy bridge, three features whose formation logically require oxidative chemistry. Correspondingly, the evd gene cluster encoding Everninomicin D encodes two monofunctional nonheme iron, α-ketoglutarate dependent oxygenases and one bifunctional enzyme with an Nterminal methyltransferase domain and a C-terminal oxygenase domain. To investigate whether the activities of these domains are linked in the bifunctional enzyme EvdMO1, we determined the structure of the N-terminal methyltransferase domain to 1.1 Å and the full-length protein to 3.35 Å resolution. Both domains of EvdMO1 adopt the canonical folds of their respective superfamilies and are connected by a short linker. Each domain's active site is oriented such that it faces away from the other domain, and there is no evidence of a channel connecting the two. Our results support EvdMO1 working as a bifunctional enzyme with independent catalytic activities.

A New Microbial Pathway for Organophosphonate Degradation Catalyzed by Two Previously Misannotated Non-Heme-Iron Oxygenases

“…There are also extant gene fusions of PhnY and PhnZ. It is unknown whether these fusions fold to be functional two-domain enzymes, but there is precedent for an active bifunctional protein with fused Fe/2OG oxygenase and methyltransferase domains . Nevertheless, the occurrence of the gene fusions supports the notions that each pairing is specific and that the pair functions together .…”

“…It is unknown whether these fusions fold to be functional two-domain enzymes, but there is precedent for an active bifunctional protein with fused Fe/ 2OG oxygenase and methyltransferase domains. 107 Nevertheless, the occurrence of the gene fusions supports the notions that each pairing is specific and that the pair functions together. 108 Furthermore, the frequent presence of a gene encoding a LysR-type transcriptional regulator upstream of the TmpA/B genes suggests that they are co-transcribed and is consistent with the biochemical evidence presented herein that their gene products constitute a degradation pathway.…”

Methyltransferase Contingencies in the Pathway of Everninomicin D Antibiotics and Analogues

“…Based on sequence similarity to the O ‐methyltransferase from the rubradirin pathway, RubN7, [21] EvdM9 encodes the O ‐methyltransferase responsible for methylating the C‐4 hydroxy of evernitrose. Finally, evdMO1 encodes a fusion protein with an N‐terminal O ‐methyltransferase with homology to aviG5 , the product of which methylates the C‐4 hydroxy of the E‐ring of avilamycin [19,22] . The C‐terminal portion of EvdMO1 is homologous to the oxidase EveO1 in M. carbonacea var.…”

“…Finally, evdMO1 encodes a fusion protein with an N-terminal O-methyltransferase with homology to aviG5, the product of which methylates the C-4 hydroxy of the E-ring of avilamycin. [19,22] The C-terminal portion of EvdMO1 is homologous to the oxidase EveO1 in M. carbonacea var. africana, wherein it is not observed as a fusion protein.…”

Orthoesters: Multiple Role Players in Organic Synthesis

“…This protective ability has made orthoesters indispensable in a range of reactions, including the synthesis of RNA oligonucleotides, plant alkaloids, and preparation of a tetracationic macrocycle that can be used as a constituent of a hydrogel . Naturally‐occurring orthoesters are essential for the pharmacological activity of certain immunosuppressive and antibiotic compounds . Previous literature reviews have mainly focused on the methods of producing orthoesters, or discussing interesting reactions of orthoesters, including the Johnson‐Claisen rearrangement .…”