View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Chemistry & Biology Article An Iterative Type I Polyketide Synthase Initiates the Biosynthesis of the Antimycoplasma Agent Micacocidin Hirokazu Kage,1 Martin F. Kreutzer,1 Barbara Wackler,2 Dirk Hoffmeister,2 and Markus Nett1,* 1Junior Research Group ‘‘Secondary Metabolism of Predatory Bacteria’’, Leibniz Institute for Natural Product Research and Infection Biology, Hans-Kno¨ ll-Institute, Beutenbergstrasse 11a, 07745 Jena, Germany 2Department of Pharmaceutical Biology at the Hans-Kno¨ ll-Institute, Friedrich Schiller Universita¨ t Jena, 07745 Jena, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2013.04.010 SUMMARY under macrolide therapy have been described (Averbuch et al., 2011; Cardinale et al., 2011; Itagaki et al., 2013). Micacocidin is a thiazoline-containing natural pro- Micacocidin is a new antibiotic that exerts strong inhibitory duct from the bacterium Ralstonia solanacearum effects in the nanomolar range against several Mycoplasma that shows significant activity against Mycoplasma species, including M. pneumoniae (Kobayashi et al., 1998). pneumoniae. The presence of a pentylphenol moiety In vivo efficacy after oral administration was demonstrated in distinguishes micacocidin from the structurally chicken that had been infected with M. gallisepticum (Kobayashi related siderophore yersiniabactin, and this residue et al., 2000). The thiazoline-containing natural product micaco- cidin is structurally closely related to the siderophore yersinia- also contributes to the potent antimycoplasma bactin (Drechsel et al., 1995), and cellular uptake studies effects. The biosynthesis of the pentylphenol moiety, suggest that it might also be involved in iron acquisition of the as deduced from bioinformatic analysis and stable producing bacterium Ralstonia solanacearum (Kreutzer et al., isotope feeding experiments, involves an iterative 2011). It is interesting that the antimycoplasma activity of mica- type I polyketide synthase (iPKS), which generates cocidin is not linked to its metal-chelating properties, as an a linear tetraketide intermediate from acyl carrier iron(III)-loaded complex exhibits the same level of activity as protein-tethered hexanoic acid by three consecu- the free ligand (Kobayashi et al., 2000). Furthermore, blockage tive, decarboxylative Claisen condensations with of the metal coordination sites by chemical modification does malonyl-coenzyme A. The final conversion into not affect the growth-inhibitory effects against Mycoplasma 6-pentylsalicylic acid depends on a ketoreductase species (Ino et al., 2001). domain within the iPKS, as demonstrated by heterol- A recent study unveiled the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes that account ogous expression in E. coli and subsequent site- for the assembly of micacocidin (Kreutzer et al., 2011). Even directed mutagenesis experiments. Our results though the domain architecture of the corresponding biosyn- unveil the early steps in micacocidin biosynthesis thesis enzymes is highly reminiscent of the yersiniabactin and illuminate a bacterial enzyme that functionally re- assembly line (Gehring et al., 1998b; Miller et al., 2002), some sembles fungal polyketide synthases. minor deviations were found to be sufficient to translate into chemical diversity (Figure 1). Both yersiniabactin and micacoci- din feature a phenolic moiety in their structures, but in the case INTRODUCTION of micacocidin, the corresponding residue is further decorated with a pentyl side chain. This structural variation apparently Mycoplasma pneumoniae is one of the leading causes of com- has important consequences in terms of biological activity munity-acquired pneumonia and other respiratory disorders. (Takeda et al., 1996), fueling the interest in its biosynthetic origin. Due to the lack of a cell wall, this bacterium is naturally resistant The phenolic moiety in yersiniabactin derives from salicylic acid, against beta-lactam and glycopeptide antibiotics. Since the which was shown to be incorporated by the stand-alone AMP- pathogen also exhibits enhanced resilience against aminoglyco- dependent ligase YbtE (Gehring et al., 1998a). In the micacocidin sides, oxazolidinones, rifampicin, and sulfonamides, therapy of pathway, the adenylation domain MicC-A1 represents the enzy- M. pneumoniae infections is restricted to a limited set of anti- matic counterpart of YbtE. However, the sequence of MicC-A1 biotics, among which macrolides are typically considered the exhibits a specificity-conferring signature that appears to be drugs of first choice (Waites and Talkington, 2004). Of note, inconsistent with the loading of an aryl carboxylic acid (Stachel- macrolides also represent the only agents that are approved haus et al., 1999; May et al., 2002). Moreover, the presence of an for the treatment of M. pneumoniae infections in children. additional type I PKS module, which is absent in the yersiniabac- Over the past decade, the prevalence of macrolide-resistant tin assembly line, was puzzling. Intrigued by these peculiarities, M. pneumoniae strains has dramatically increased (Jacobs, we set out to resolve the molecular mechanism by which the 2012), and more recently, several cases of resistance emerging pentylphenol moiety of micacocidin is formed. The knowledge 764 Chemistry & Biology 20, 764–771, June 20, 2013 ª2013 Elsevier Ltd All rights reserved Chemistry & Biology An iPKS Initiates Micacocidin Assembly Figure 1. Modular Biosynthesis Enzymes for the Production of Micacocidin (A) Domain architecture of the micacocidin (Mic) assembly line. Domains that likely account for the structural differences to yersiniabactin (Ybt) are highlighted in blue. Domain notation: A, adenylation; ACP, acyl carrier protein; KS, keto- synthase; AT, acyl transferase; KR, ketor- eductase; C, condensation; MT, SAM-dependent methyltransferase; PCP, peptidyl carrier protein; TE, thioesterase. (B) Chemical structures of micacocidin and yersiniabactin. Townsend, 2010). In the second sce- nario, we proposed the loading of an oxygenated decanoic acid, which would undergo a single decarboxylative thio- Claisen condensation with malonyl-CoA to give a C12 diketide. Again, an aldol- on the biosynthesis will, in the long term, help manipulate pro- type reaction could cyclize the thioester-bound intermediate, duction and generate structural derivatives of this promising which would then be further oxidized to PSA. antibiotic. In order to distinguish between the two routes, the loading module of MicC was heterologously produced and purified RESULTS AND DISCUSSION from Escherichia coli KRX as an N-terminal hexahistidine fusion via the pET28a(+)-derived expression plasmid pHiK007 (Fig- Identification of the Starter Unit in Micacocidin ure S2; for primer sequences, see Table S1). The substrate Biosynthesis preference of the recombinant protein was tested in the ATP- Feeding studies with stable isotope precursors revealed that the [32P]-pyrophosphate exchange assay, as previously described metabolic origin of the pentylphenol moiety is dissimilar from the (Kreutzer et al., 2012). This approach revealed a clear preference salicylate building block in yersiniabactin. While the latter derives for hexanoic acid, whereas the other possible starter molecule, from shikimate metabolism (Gehring et al., 1998a), the incorpo- decanoic acid, was not activated at relevant levels (Figure 2). ration pattern of 13C-labeled acetate into micacocidin was in Further testing showed a narrow substrate tolerance of the full agreement with a polyketide origin (Figure S1 available enzyme. While pentanoic acid and heptanoic acid could still be online). It paralleled that of the plant metabolite olivetolic acid converted into their corresponding adenylate products with (4-hydroxy-6-pentylsalicylic acid), which is an important inter- low efficiency, octanoic acid already failed to give an appreciable mediate in the pathway to cannabinoids (Fellermeier et al., response. A subsequent feeding study in R. solanacearum with 2001). The biosynthesis of olivetolic acid involves a type III [1-13C]hexanoic acid led to an increased incorporation of label PKS, which forms a linear tetraketide from hexanoyl-coenzyme at C-3 of micacocidin (Figure S1), ultimately confirming this A (CoA) and three molecules of malonyl-CoA (Taura et al., short-chain fatty acid as the biosynthetic starter unit. 2009). Conversion of the tetraketide into olivetolic acid was not solved until recently, when a cyclase was found to catalyze the Heterologous Production of PSA in Escherichia coli concluding intramolecular aldol condensation (Gagne et al., To investigate whether the PKS module of MicC catalyzes the 2012). Based upon the results from the feeding studies in proposed iterative C2 chain elongation of primed hexanoic R. solanacearum, an analogous mechanism for the formation acid, a truncated enzyme including the loading and the first of 6-pentylsalicylic acid (PSA) would be conceivable, yet the extender module (A1-ACP-KS-AT-KR-ACP) was recombinantly entire genome of the micacocidin producer is devoid of genes produced. The assembly of the respective expression vector, for type III PKSs (Salanoubat et al., 2002). Therefore, we envis- pHiK008, was accomplished by l Red-mediated in vivo cloning aged two alternative scenarios that could give rise to PSA and (Zhang et al., 2000; Figure S3). For gene expression, pHiK008 that