DOI: 10.1002/cbic.201000200 Pacidamycin Biosynthesis: Identification and Heterologous Expression of the First Uridyl Peptide Antibiotic Gene Cluster Emma J. Rackham, [a] Sabine Grüschow, [a] Amany E. Ragab, [a] Shilo Dickens, [b] and Rebecca J. M. Goss* [a] Introduction The worrying development of resistance to commercial antibi- otics, coupled to the dearth of new antibiotics reaching the market, provides the impetus for research aimed at the discov- ery of novel antibiotic compounds. The pacidamycins, pro- duced by Streptomyces coeruleorubidus (Figure 1) possess novel structural features and exhibit an unusual selectivity against Pseudomonas aeruginosa. [1–3] They belong to a family of closely related uridyl peptide antibiotics that also includes the murei- domycins, napsamycins and sansanmycins (Figure 1). [4–6] The uridyl peptide antibiotics exhibit a clinically unexploited mode of action against translocase I. Identification of the biosynthetic gene cluster will allow the generation of new designer ana- logues of these attractive antibiotics by combinatorial biosyn- thesis. The activity of this class of compounds stems from inhibition of phospho-N-acetyl-muramylpentapeptide translocase I, an essential enzyme involved in bacterial cell-wall biosynthesis. [7] Translocase I catalyses the transfer of the phospho-N-acetyl- muramylpentapeptide precursor onto the undecaprenyl phos- phate carrier to yield the first membrane-bound intermediate in cell-wall biosynthesis. A puzzling aspect of uridyl peptide antibiotics is their almost exclusive antipseudomonal activity. The observed selectivity is not due to differences in the struc- ture of translocase I between species: in vitro experiments with mureidomycins demonstrated effective inhibition of trans- locase I from both Escherichia coli and Staphylococcus aureus. [8–10] The novel scaffold found in the pacidamycin family is com- posed of a uridine nucleoside with an atypical 3’-deoxyribose aminosugar component. The nucleoside is linked through a unique exocyclic enamide to the tetra- (or penta-)pseudopep- tide. The peptide backbone contains a rare (2S,3S)-diaminobu- tyric acid (DABA) residue and internal ureido moiety that each reverse the direction of the peptide chain. Furthermore, the majority of the pacidamycins incorporate the rare meta-tyro- sine residue, and the amino terminus can be capped by an un- usual bicyclic heterocycle. [1, 7] The total synthesis of dihydropacidamycin D (Figure 1) was accomplished in 2001, and the product displayed antipseudo- monal activity comparable to that of pacidamycin D; this re- vealed that the enamide is not necessary for translocase I inhibition. [10, 11] Instead, the uridine nucleoside is thought to mimic the enzyme’s natural UDP substrate and bind in the translocase I active site. It is therefore not surprising that changes to the sugar or base of the uridine nucleoside are only poorly tolerated in terms of enzyme inhibition. [11, 12] The amino terminus of the pacidamycins is proposed to compete for the Mg 2 + binding site of translocase I; a peptide chain of appropriate length and conformation linking the amino termi- nus to the nucleoside portion is required for optimum activi- ty. [13–15] Conversely, certain variations to the amino acid resi- The pacidamycins are antimicrobial nucleoside antibiotics pro- duced by Streptomyces coeruleorubidus that inhibit transloca- se I, an essential bacterial enzyme yet to be clinically targeted. The novel pacidamycin scaffold is composed of a pseudopep- tide backbone linked by a unique exocyclic enamide to an atypical 3’-deoxyuridine nucleoside. In addition, the peptidyl chain undergoes a double inversion caused by the incorpora- tion of a diamino acid residue and a rare internal ureido moiety. The pacidamycin gene cluster was identified and se- quenced, thereby providing the first example of a biosynthetic cluster for a member of the uridyl peptide family of antibiotics. Analysis of the 22 ORFs provided an insight into the biosynthe- sis of the unique structural features of the pacidamycins. Het- erologous expression in Streptomyces lividans resulted in the production of pacidamycin D and the newly identified pacida- mycin S, thus confirming the identity of the pacidamycin bio- synthetic gene cluster. Identification of this cluster will enable the generation of new uridyl peptide antibiotics through com- binatorial biosynthesis. The concise cluster will provide a useful model system through which to gain a fundamental understanding of the way in which nonribosomal peptide syn- thetases interact. [a] E. J. Rackham, Dr. S. Grüschow, A. E. Ragab, Dr. R. J. M. Goss School of Chemistry, University of East Anglia Earlham Road, Norwich NR4 7TJ (UK) Fax: (+ 44) 1603-592-003 [b] Dr. S. Dickens Department of Biochemistry, University of Cambridge Tennis Court Road, Cambridge CB2 1GA (UK) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000200. 1700  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2010, 11, 1700 – 1709