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The Structural Biology of Patellamide Biosynthesis

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The Structural Biology of Patellamide Biosynthesis Available online at www.sciencedirect.com ScienceDirect The structural biology of patellamide biosynthesis 1 1 2,3 Jesko Koehnke , Andrew F Bent , Wael E Houssen , 1 2 1 Greg Mann , Marcel Jaspars and James H Naismith The biosynthetic pathways for patellamide and related natural the pharmaceutical armory for treating diseases ranging products have recently been studied by structural biology. from bacterial infection to cancer to immune suppression These pathways produce molecules that have a complex [4]. Synthetic biology promises, amongst other deliver- framework and exhibit a diverse array of activity due to the ables, the ability to tailor enzymes in biosynthetic pathways variability of the amino acids that are found in them. As these to create natural product variants with desirable properties molecules are difficult to synthesize chemically, exploitation of in the quantities required for drug development [5]. their properties has been modest. The patellamide pathway involves amino acid heterocyclization, peptide cleavage, Peptide based natural products are particularly attractive peptide macrocyclization, heterocycle oxidation and from a chemical point of view; amino acids share a common epimerization; closely related products are also prenylated. standard in connectivity (the amide bond) with an almost Enzyme activities have been identified for all these infinitely configurable element (the side chain). One can transformations except epimerization, which may be thus ‘dial’ in chemical and structural properties into a spontaneous. This review highlights the recent structural and shared basic design. Crucially, in the same way as no mechanistic work on amino acid heterocyclization, peptide two proteins need to share any biological property, pep- cleavage and peptide macrocyclization. This work should help tides by virtue of their different side chains can have in using the enzymes to produce novel analogs of the natural divergent properties. The cyanobactin family of riboso- products enabling an exploitation of their properties. mally synthesized and post-translationally modified pep- Addresses tides (RiPPs) are peptide macrocycles that exemplify this 1 BSRC, North Haugh, The University, St Andrews KY16 9ST, UK diversity, ranging from six to over 20 residues, with diver- 2 Marine Biodiscovery Centre, Department of Chemistry, University of gent sequences and very different biological properties Aberdeen, Meston Walk, Aberdeen AB24 3UE, UK 3 including P-gp inhibition, cytotoxicity, immunomodula- Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 tion, antifungal, antibacterial and antiviral properties [6]. 2ZD, UK All authors contributed equally to the review. Macrocyclic peptides are particularly appealing as they are intrinsically resistant to protease degradation and several Corresponding authors: Jaspars, Marcel ([email protected]) and cross membranes [7]. There is significant interest in their Naismith, James H ([email protected]) biosynthesis with a view to exploiting this class of molecule for novel drugs [8 ,9,10]. In this review, we use the example Current Opinion in Structural Biology 2014, 29:112–121 of the patellamide (an eight-residue macrocyclic cyano- bactin) [11] to structure our discussion. In this pathway a This review comes from a themed issue on Catalysis and regulation single ribosomal seventy one-residue precursor peptide, Edited by James H Naismith and Emily J Parker PatE, gives rise to two eight-residue macrocycles; patella- For a complete overview see the Issue and the Editorial mide A and patellamide C [11] (Figure 1a). PatE contains Available online 25th November 2014 two eight-residue core peptides and each is converted to http://dx.doi.org/10.1016/j.sbi.2014.10.006 the corresponding patellamides (Figure 1b) by enzyme action. Within PatE each core peptide is flanked by a 0959-440X/# 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecom- conserved five-residue protease signature (N-terminal), a mons.org/licenses/by/3.0/). conserved three-residue macrocyclization signature (C- terminal) and in addition PatE has a thirty seven-residue leader peptide at N-terminus [11] (Figure 1a). During synthesis the peptide bonds between the core peptide Introduction and its flanking regions must be cut and the ends of the Small molecules with potent biological properties are core peptide joined (Figure 1b). The final products contain commonly isolated from bacteria. Analysis of well estab- oxazolines (derived from the heterocyclization of serine/ lished rules for anthropogenic pharmaceuticals [1] indicates threonine) and thiazoles (oxidized form of thiazoline which that in comparison, natural products tend to have more is derived from the heterocyclization of cysteine) stereo centres and nitrogen atoms but are, in general, not (Figure 1b). The two residues adjacent to the thiazoles wildly different [2,3]. Natural products that have some are epimerized from an L-configuration to D-configuration undesirable properties as medicines, but they can be (Figure 1b). The functions of the enzymes that convert improved by chemical modification. Natural products, PatE into patellamides were first assigned by sequence including modified variants, are a major component in analysis (Figure 1c) [11]. There are many closely related Current Opinion in Structural Biology 2014, 29:112–121 www.sciencedirect.com Natural product biosynthesis Koehnke et al. 113 Figure 1 (a) patA patB patC patD patE patF patG (X)37 GLEAS VTACITFC AYD GVEPS ITVCISVCAYD GE Leader Protease Core Macro Core site peptide signature peptide Patellamide C Patellamide A (b) Herocyclization / cyclic dehydration Epimerization at Cα O R3 R4 Prenylation S Oxidation N S O N H N HN O N Proteolysis O NH Macrocyclization NH HN O O O O R O O 1 HN O N H N N S O N R2 O Patellamide A: R =CHMeEt; R ,R =CHMe ; R =H 1 2 4 2 3 Patellin Patellamide C: R1 =CHMe2; R2,R3=Me; R4=CH2Ph (c) PatA protease DUF PatD heterocyclase / cyclodehydratase PatF prenyl transferase PatG oxidase macrocyclase DUF Current Opinion in Structural Biology (a) The patellamide gene cluster contains genes patA to patG, coding for proteins PatA to PatG. The PatE protein contains: (1) a 37 residue leader and (2) two core peptides, which are processed to give patellamides C and A [11]. The core peptides are flanked at their N and C termini; (b) the final natural product patellamides C and A showing the chemical transformations during post-translational processing. The closely related patellin which is prenylated is shown; (c) the enzymes which tailor the core peptide. PatG encodes two functions and in addition has a DUF. PatA encodes the protease and also posses the same DUF. pathways in other marine organisms, such as the trunka- threonine residues, too) within the context of the core mide and patellin (Figure 1b) biosynthetic pathway, that peptide(s), to create thiazolines (or oxazolines) utilise biosynthetic enzymes which are very similar to those (Figure 1b) and eliminate water [12]. Patellamides A and of the patellamide pathway [6,9,11]. Moreover, many of the C contain four heterocycles each and these five membered chemical reactions that are catalyzed by enzymes in the rings profoundly change the chemistry and flexibility of the patellamide pathway occur in the biosynthesis of other peptide [13]. Their selective introduction is a powerful tool natural products that are unrelated to cyclic peptides. We to tune the molecular shape and activity of not only peptide herein discuss insights from these other pathways under macrocycles, but also linear peptide natural product the corresponding chemical transformations observed in families such as the antibacterial microcin peptides [14]. the patellamide system. The microcin ‘Trojan horse’ antibiotic peptide MccC7 has a C-terminal phosphoramidate that is linked to adenosine. Heterocyclization During the biosynthesis of the peptide an intermediate The heterocyclase (or cyclodehydratase) class of enzymes with a C-terminal five membered succinamide ring is modify cysteine residues (and in some cases serine and synthesized by heterocyclization of the C-terminal Asn www.sciencedirect.com Current Opinion in Structural Biology 2014, 29:112–121 114 Catalysis and regulation residue [15]. The structure of the enzyme responsible, seen in other adenylating enzymes [23 ]. An adenylase MccB, was revealed to have two domains. The larger of rather than a kinase mechanism was proposed as the most the two (the C-terminal) adopts a classic adenylase super- likely for TruD with the data in hand [23 ]. family fold whilst the fold of the smaller (N-terminal) was novel [15]. MccB activates the substrate peptide carboxy TruD was shown to have a preferred order of reactivity, terminus by first converting it to an adenylate then the proceeding from the C-terminus [23 ]. The processivity amide of the side chain of Asn displaces AMP to give the of the enzyme required the leader peptide to be attached succinamide five membered ring. MccB catalyzes a second to the core peptide and similar observations had pre- adenylation reaction that adds adenosine to the peptide. viously been made for the BalhC/D system [24 ,25 ]. All Domain 1 of MccB acts as a clamp holding the peptide the heterocyclase enzymes are promiscuous and tolerate substrate for processing. More recently thiazole/thiazoline- diversity in chemical and structural nature of amino acids, containing (modified) microcins [16], collectively known as which
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