ABSTRACT
The rapid development of antibiotic resistance, especially among difficult-to-treat Gram-negative bacteria, is recognized as a serious and urgent threat to public health. The detection and characterization of novel resistance mechanisms are essential to better predict the spread and evolution of antibiotic resistance. Corramycin is a novel and modified peptidic antibiotic with activity against several Gram-negative pathogens. We demonstrate that the kinase ComG, part of the corramycin biosynthetic gene cluster, phosphorylates and thereby inactivates corramycin, leading to the resistance of the host. Remarkably, we found that the closest structural homologues of ComG are aminoglycoside phosphotransferases; however, ComG shows no activity toward this class of antibiotics. The crystal structure of ComG in complex with corramycin reveals that corramycin adopts a ß-hairpin-like structure and allowed us to define the changes leading to a switch in substrate from sugar to peptide. Bioinformatic analyses suggest a limited occurrence of ComG-like proteins, which along with the absence of cross-resistance to clinically used drugs positions corramycin as an attractive antibiotic for further development.
Subject(s)
Anti-Bacterial Agents , Gram-Negative Bacteria , Anti-Bacterial Agents/chemistry , Gram-Negative Bacteria/metabolism , Kanamycin Kinase/chemistry , Kanamycin Kinase/genetics , Kanamycin Kinase/metabolism , PeptidesABSTRACT
Herein, we describe the myxobacterial natural product Corramycin isolated from Corallococcus coralloides. The linear peptide structure contains an unprecedented (2R,3S)-γ-N-methyl-ß-hydroxy-histidine moiety. Corramycin exhibits anti-Gram-negative activity against Escherichia coli (E.â coli) and is taken up via two transporter systems, SbmA and YejABEF. Furthermore, the Corramycin biosynthetic gene cluster (BGC) was identified and a biosynthesis model was proposed involving a 12-modular non-ribosomal peptide synthetase/polyketide synthase. Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule's absolute configuration. Importantly, intravenous administration of 20â mg kg-1 of Corramycin in an E.â coli mouse infection model resulted in 100 % survival of animals without toxic side effects. Corramycin is thus a promising starting point to develop a potent antibacterial drug against hospital-acquired infections.
Subject(s)
Anti-Bacterial Agents , Escherichia coli , Mice , Animals , Anti-Bacterial Agents/chemistry , Polyketide Synthases , Multigene FamilyABSTRACT
Tilvalline is a pyrrolo[4,2]benzodiazepine derivative produced by the pathobiont Klebsiella oxytoca and is the causative toxin in antibiotic associated hemorrhagic colitis (AAHC). Heterologous expression of the tilivalline biosynthetic gene cluster along with in vitro reconstitution of the respective NRPS (NpsA, ThdA, NpsB) was employed to reveal a nonenzymatic indole incorporation via a spontaneous Friedel-Crafts-like alkylation reaction. Furthermore, the heterologous system was used to generate novel tilivalline derivatives by supplementation of respective anthranilate and indole precursors. Finally, it could be shown that salicylic and acetylsalicylic acid inhibit the biosynthesis of tilivalline in K. oxytoca liquid culture, presumably by blocking the peptidyl carrier protein ThdA, pointing toward a potential application in combination therapy to prevent or alleviate the symptoms of AAHC.
Subject(s)
Benzodiazepinones/metabolism , Enterocolitis, Pseudomembranous/drug therapy , Klebsiella oxytoca/pathogenicity , Benzodiazepinones/chemical synthesis , Benzodiazepinones/chemistry , Cytotoxins/chemical synthesis , Enterocolitis, Pseudomembranous/etiology , Indoles/metabolism , Klebsiella oxytoca/chemistry , Klebsiella oxytoca/metabolism , Multigene Family , Virulence Factors/genetics , Virulence Factors/metabolism , ortho-Aminobenzoates/metabolismABSTRACT
In vitro reconstitution and biochemical analysis of natural product biosynthetic pathways remains a challenging endeavor, especially if megaenzymes of the nonribosomal peptide synthetase (NRPS) type are involved. In theory, all biosynthetic steps may be deciphered using mass spectrometry (MS)-based analyses of both the carrier protein-coupled intermediates and the free intermediates. We here report the "total biosynthesis" of the pyrrolo[4,2]benzodiazepine scaffold tomaymycin using an in vitro reconstituted NRPS system. Proteoforms were analyzed by liquid chromatography (LC)-MS to decipher every step of the biosynthesis on its respective megasynthetase with up to 170 kDa in size. To the best of our knowledge, this is the first report of a comprehensive analysis of virtually all chemical steps involved in the biosynthesis of nonribosomally synthesized natural products. The study includes experiments to determine substrate specificities of the corresponding A-domains in competition assays by analyzing the adenylation step as well as the transfer to the respective carrier protein domain.
Subject(s)
Benzodiazepines/chemistry , Peptide Synthases/metabolism , Pyrroles/chemistry , Benzodiazepinones/chemistry , Benzodiazepinones/metabolism , Models, Molecular , Peptide Synthases/chemistry , Protein Domains , Substrate SpecificityABSTRACT
The nocazines are a newly defined family of antibacterial and cytotoxic cyclic dipeptides produced by different actinobacterial species. Here, we identify a nocazine biosynthetic gene cluster in Nocardiopsis dassonvillei and describe the elucidation of the biosynthetic pathway leading to the nocazine family members nocazine E and XR334. Diketopiperazine (DKP) formation is carried out by a tRNA-dependent cyclodipeptide synthase (CDPS) showing an unknown product profile, while tailoring of the DKP-scaffold is achieved through the combined and combinatorial action of a cyclodipeptide oxidase and two distinct SAM-dependent O-/N-methyltransferases. Our results help to illuminate the biosynthetic logic resulting in the structural diversity of the nocazine family and set the stage for exploring the biological function of modified cyclic dipeptides as possible mediators of host-pathogen and host-parasite interactions.
Subject(s)
Dipeptides/metabolism , RNA, Transfer/metabolism , Actinomycetales/classification , Actinomycetales/genetics , Actinomycetales/metabolism , Amino Acid Sequence , Diketopiperazines/chemistry , Diketopiperazines/metabolism , Dipeptides/chemistry , Genome, Bacterial , Methyltransferases/metabolism , Molecular Sequence Data , Multigene Family , Peptide Synthases/metabolism , Phylogeny , Piperazines/chemistry , Piperazines/metabolism , Sequence AlignmentABSTRACT
A large number of bioactive natural products containing a 2,5-diketopiperazine (DKP) moiety have been isolated from various microbial sources. Especially tryptophan-containing cyclic dipeptides (CDPs) show great structural and functional diversity, while little is known about their biosynthetic pathways. Here, we describe the bioinformatic analysis of a cyclodipeptide synthase (CDPS)-containing gene cluster from Actinosynnema mirum spanning 2.9 kb that contains two putative DKP-modifying enzymes. We establish the biosynthetic pathway leading to two methylated ditryptophan CDPs through in vivo and in vitro analyses. Our studies identify the first CDPS (Amir_4627) that shows high substrate specificity synthesizing only one main product, cyclo(Trp-Trp) (cWW). It is the first member of the CDPS family that can form ditryptophan DKPs and the first prokaryotic CDPS whose main product constituents differ from the four amino acids (Phe, Leu, Tyr, and Met) usually found in CDPS-dependent CDPs. We show that after cWW formation a S-adenosyl-l-methionine-dependent N-methyltransferase (Amir_4628) conducts two successive methylations at the DKP-ring nitrogens and additionally show that it is able to methylate four other phenylalanine-containing CDPs. This makes Amir_4628 the first identified DKP-ring-modifying methyltransferase. The large number of known modifying enzymes of bacterial and fungal origin known to act upon Trp-containing DKPs makes the identification of a potent catalyst for cWW formation, encoded by a small gene, valuable for combinatorial in vivo as well as chemoenzymatic approaches, with the aim of generating derivatives of known CDP natural products or entirely new chemical entities with potentially improved or new biological activities.