Abstract
Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.
Highlights
Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria
Due to the unusual structure of kistamicin when compared with typical type I–IV glycopeptide antibiotics (GPAs), we initially sequenced the genome of Actinomadura parvosata subsp. kistnae
A phylogenetic analysis of the Oxy enzymes from kistamicin biosynthesis revealed that these fall into the OxyA (KisN) and OxyC (KisO) families and do not cluster with OxyB, the first enzyme involved in cyclisation cascade of the Type I-IV GPAs (Supplementary Fig. 2)
Summary
Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. Due to the clinical utility of GPAs and the challenges associated with the synthesis of these complex molecules, significant research efforts have been made to understand the cyclisation process of GPAs from both an in vivo[3,10,11] and in vitro standpoint[9,12,13,14,15] These studies have revealed that each Oxy enzyme is responsible for the installation of one crosslink at the heptapeptide stage (Fig. 1b) and that a specific order of activity exists (OxyB, (±OxyE), OxyA, OxyC). Given the importance of the GPA cyclisation cascade, identifying new members of related biosynthetic machineries is key to understanding the biocatalytic potential of these powerful catalysts: for this reason, there is great interest in an unusual class of GPAs—known as Type V GPAs— that include the representative members complestatin[16,17] and kistamicin[18,19]. Type V GPAs display divergent activity when compared to Type I–IV GPAs, including antiviral activity for both kistamicin[19] and complestatin[20,21] as well as other types of potential antibacterial activity[22]; this clearly makes such atypical GPAs of great interest for diversification of the activity of GPAs
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