RESUMEN
Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates.
Asunto(s)
Calcio , Pared Celular , Péptidos , Uridina Difosfato Ácido N-Acetilmurámico , Pared Celular/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/química , Calcio/metabolismo , Péptidos/farmacología , Péptidos/metabolismo , Péptidos/química , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/metabolismo , Uridina Difosfato Ácido N-Acetilmurámico/química , Microscopía de Fuerza Atómica , Antibacterianos/farmacología , Antibacterianos/química , Espectroscopía de Resonancia Magnética , Unión ProteicaRESUMEN
Lipid II is an essential glycolipid found in bacteria. Accessing this valuable cell wall precursor is important both for studying cell wall synthesis and for studying/identifying novel antimicrobial compounds. Herein, we describe optimizations to the modular chemical synthesis of lipid II and unnatural analogues. In particular, the glycosylation step, a critical step in the formation of the central disaccharide unit (GlcNAc-MurNAc), was optimized. This was achieved by employing the use of glycosyl donors with diverse leaving groups. The key advantage of this approach lies in its adaptability, allowing for the generation of a wide array of analogues through the incorporation of alternative building blocks at different stages of synthesis.
RESUMEN
Undecaprenol-containing glycolipids (UCGs) are essential precursors of bacterial glycopolymers and glycoproteins. We report a novel semi-synthetic strategy to prepare labelled UCGs directly from undecaprenol. This one-size-fits-all approach offers a concise and efficient method for obtaining labelled-UCGs, which will allow new mechanistic studies and inhibitor screens to be performed on novel antibiotic targets.
Asunto(s)
Antibacterianos/química , Terpenos/química , Antibacterianos/síntesis química , Antibacterianos/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Glucolípidos/química , Bacterias Grampositivas/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Plantas/química , Plantas/metabolismo , Terpenos/síntesis química , Terpenos/metabolismoRESUMEN
The antimalarial agent cladosporin is a nanomolar inhibitor of the Plasmodium falciparum lysyl-tRNA synthetase, and exhibits activity against both blood- and liver-stage infection. Cladosporin can be isolated from the fungus Cladosporium cladosporioides, where it is biosynthesized by a highly reducing (HR) and a non-reducing (NR) iterative typeâ I polyketide synthase (PKS) pair. Genome sequencing of the host organism and subsequent heterologous expression of these enzymes in Saccharomyces cerevisiae produced cladosporin, confirming the identity of the putative gene cluster. Incorporation of a pentaketide intermediate analogue indicated a 5+3 assembly by the HR PKS Cla2 and the NR PKS Cla3 during cladosporin biosynthesis. Advanced-intermediate analogues were synthesized and incorporated by Cla3 to furnish new cladosporin analogues. A putative lysyl-tRNA synthetase resistance gene was identified in the cladosporin gene cluster. Analysis of the active site emphasizes key structural features thought to be important in resistance to cladosporin.
Asunto(s)
Antimaláricos/metabolismo , Isocumarinas/química , Sintasas Poliquetidas/metabolismo , Isocumarinas/metabolismoRESUMEN
Iterative typeâ I polyketide synthases (PKSs) from fungi are multifunctional enzymes that use their active sites repeatedly in a highly ordered sequence to assemble complex natural products. A phytotoxic macrolide with anticancer properties, 10,11-dehydrocurvularin (DHC), is produced by cooperation of a highly reducing (HR) iterative PKS and a non-reducing (NR) iterative PKS. We have identified the DHC gene cluster in Alternaria cinerariae, heterologously expressed the active HR PKS (Dhc3) and NR PKS (Dhc5) in yeast, and compared them to corresponding proteins that make DHC in Aspergillus terreus. Phylogenetic analysis and homology modeling of these enzymes identified variable surfaces and conserved motifs that are implicated in product formation.
Asunto(s)
Alternaria/enzimología , Aspergillus/enzimología , Sintasas Poliquetidas/metabolismo , Zearalenona/análogos & derivados , Alternaria/genética , Aspergillus/genética , Dominio Catalítico , Familia de Multigenes , Filogenia , Sintasas Poliquetidas/clasificación , Sintasas Poliquetidas/genética , Estructura Terciaria de Proteína , Zearalenona/biosíntesis , Zearalenona/químicaRESUMEN
Biosynthesis of bioactive natural products frequently features oxidation at multiple sites. Starting from a relatively reduced chemical scaffold that is assembled by controlled polymerization of small precursors, for example, acetate or amino acids, a diverse range of redox reactions can generate very complex and highly oxygenated structures. Their formation often involves C-H activation reactions catalyzed by oxygenase enzymes, either monooxygenases or dioxygenases. The former category includes the cytochrome P450s and flavin-dependent oxygenases, whereas examples of the latter are the non-heme iron α-ketoglutarate-dependent oxygenases. Oxygenases can catalyze a plethora of reactions ranging from hydroxylations and epoxidations to dehydrogenations, cyclizations, and rearrangements. The specific transformations are usually possible only with the use of these enzymatic catalysts. Aside from the ability of oxygenases to specifically oxidize unactivated carbon skeletons, some have recently been demonstrated to possess a fascinating ability to catalyze multiple reactions in a highly ordered fashion at different sites starting with a single substrate molecule. In the past, oxygenases associated with secondary metabolite pathways were considered to be highly regio-, stereo-, and substrate specific, with one oxidizing enzyme encoded in the gene cluster corresponding to one oxidation location in the natural product itself. However, it is becoming progressively clear that this "one oxygenase, one oxidation site" relationship is not necessarily a valid assumption. Multifunctional oxidases are known to occur in higher plants, fungi, and bacteria. Natural product gene clusters that contain multifunctional oxidase enzymes are responsible for production of lovastatin (a cholesterol-lowering agent and precursor to simvastatin), scopolamine (an anticholinergic drug), and cytochalasin E (an angiogenesis inhibitor), among many others. As opposed to simply being substrate promiscuous, these enzymes show very high substrate specificity and catalyze several oxidative reactions in a single pathway, with each oxidation being a prerequisite for the next. The basis for their specificity and highly ordered sequence is not yet well understood. In the lovastatin pathway, LovA is a cytochrome P450 that introduces a double bond and a hydroxyl group. H6H is an α-ketoglutarate-dependent oxygenase that hydroxylates (-)-atropine and then closes the newly introduced oxygen onto a neighboring methylene to generate the epoxide of scopolamine. CcsB is a flavin-dependent Baeyer-Villigerase that converts a ketone to a carbonate by double oxidation, a reaction not possible without enzymes. Recent crystallographic studies of other multifunctional oxygenases, such as AurH, a cytochrome P450 from Streptomyces thioluteus involved in aureothin biosynthesis, have indicated a steric switch mechanism. After the initial hydroxylation reaction catalyzed by AurH, the enzyme is thought to undergo a substrate-induced conformational change. In this Account, advances in our knowledge of these fascinating multifunctional enzymes and their potential will be explored.