The uridyl peptide antibiotics (UPAs), of which pacidamycin is a member, have a clinically unexploited mode of action and an unusual assembly. Perhaps the most striking feature of these molecules is the biosynthetically unique 3’deoxyuridine that they share. This moiety is generated by an unusual, small and monomeric dehydratase, Pac13, which catalyses the dehydration of uridine-5’-aldehyde. Here we report the structural characterisation of Pac13 with a series of ligands, and gain insight into the enzymeQs mechanism demonstrating that H42 is critical to the enzymeQs activity and that the reaction is likely to proceed via an E1cB mechanism. The resemblance of the 3’-deoxy pacidamycin moiety with the synthetic anti-retrovirals, presents a potential opportunity for the utilisation of Pac13 in the biocatalytic generation of antiviral compounds. Nucleic acids play a central role in nature and modified nucleosides are present in a wide range of anti-viral, anticancer drugs and antibiotics. Though a variety of naturally occurring nucleic acid analogues exist, few include modifications to the ribose or deoxyribose ring. The uridyl peptide antibiotics (UPAs) pacidamycin, naspamycin, mureidomycin and sansanmycin, attract much attention with a clinically unexploited mode of action and an unusual biosynthetic assembly. Intriguingly, the (UPAs) contain a biosynthetically distinct 3’-deoxyuridine that resembles the synthetic antiretrovirals such as stavudine 4, abacavir 5 (Figure 1) and the cytotoxic natural product cordycepin 6, the biosynthesis of which has not yet been determined. A detailed mechanistic understanding of the individual enzymes employed in the generation of the 3’-deoxyuridine core is required in order to facilitate their future biotransformative potential. In pacidamycin 3, biosynthesis the 3’-deoxy moiety is mediated by Pac13, an enzyme found to catalyze the key dehydration of uridine-5’-aldehyde 1 to form 3’-deoxy-3’,4’didehydrouridine-5’-aldehyde 2. Previously proposed as a dehydratase investigations into the structure, kinetics and mechanism of this unusual enzyme remained to be performed. In stark contrast to most characterised dehydratases, Pac13 is small (121 aa), monomeric, co-factor independent and utilises a non-activated nucleoside, rather than a free monosaccharide, as a substrate. The biosynthetic uniqueness coupled to the potential synthetic utility inspired us to investigate Pac13Qs structure and mechanism. This is the first mechanistic study of the formation of the 3’deoxynucleosides in natural product biosynthesis. We demonstrate that not only is Pac13 unusually small, monomeric and cofactor independent, but that it is also mechanistically distinctive. Dehydratases are important enzymes in primary and secondary metabolism and have been shown to mediate catalysis via a variety of mechanisms including metaldependent, acid-base, radical and covalent mechanisms which we summarise in the Supporting Information (SI), Figure 1. Medicinally relevant compounds containing 3’-deoxy-nucleoside moieties: A) biogenesis of the pacidamycin nucleoside motif: Pac13 catalyses dehydration of uridine-5’-aldehyde 1 to form 3’-deoxy3’,4’-didehydrouridine-5’-aldehyde 2 ; B) pacidamycin, an uridyl peptide antibiotic (highlighted: the pacidamycin nucleoside core, the characteristic 3’-deoxy uridine); C) stavudine 4 and abacavir 5, widely used antivirals and cordycepin 6, cytotoxic natural product. [*] F. Michailidou, Dr. A. F. Bent, Prof. J. H. Naismith, Dr. S. V. Sharma,