P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds dehydrogenation of a specific acylated dipeptide substrate.

WS9326A is a peptide antibiotic containing a highly unusual -methyl--2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by (P450) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450-mediated ,-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450 is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate ()-2-pent-1'-enyl-cinnamoyl-Thr--Me-Tyr.

The Cytochrome P450 OxyA from the Kistamicin Biosynthesis Cyclization Cascade is Highly Sensitive to Oxidative Damage.

Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenases that utilize a cysteine thiolate-ligated heme moiety to perform a wide range of demanding oxidative transformations. Given the oxidative power of the active intermediate formed within P450s during their active cycle, it is remarkable that these enzymes can avoid auto-oxidation and retain the axial cysteine ligand in the deprotonated-and thus highly acidic-thiolate form. While little is known about the process of heme incorporation during P450 folding, there is an overwhelming preference for one heme orientation within the P450 active site.

Chemical probes reveal the timing of early chlorination in vancomycin biosynthesis.

Glycopeptides such as vancomycin are antibiotics of last resort whose biosynthetic pathways still hold undefined details. Chemical probes were used to capture biosynthetic intermediates generated in the nonribosomal peptide formation of vancomycin in vivo. The putative intercepted intermediates were characterised via HR-LC-MS.

A Chemoenzymatic Approach to the Synthesis of Glycopeptide Antibiotic Analogues.

Glycopeptide antibiotics (GPAs) are important antibiotics that are highly challenging to synthesise due to their unique and heavily crosslinked structure. Given this, the synthetic production and diversification of this key compound class remains impractical. Furthermore, the possibility of biosynthetic reengineering of GPAs is not yet feasible since the selectivity of the biosynthetic crosslinking enzymes for altered substrates is largely unknown.

Allelic association with ankylosing spondylitis fails to correlate with human leukocyte antigen B27 homodimer formation.

Expression of human leukocyte antigen (HLA)-B27 is strongly associated with predisposition toward ankylosing spondylitis (AS) and other spondyloarthropathies. However, the exact involvement of HLA-B27 in disease initiation and progression remains unclear. The homodimer theory, which proposes that HLA-B27 heavy chains aberrantly form homodimers, is a central hypothesis that attempts to explain the role of HLA-B27 in disease pathogenesis.

Enzymatic Cascade To Evaluate the Tricyclization of Glycopeptide Antibiotic Precursor Peptides as a Prequel to Biosynthetic Redesign.

Natural products are the greatest source of antimicrobial agents, although their structural complexity often renders synthetic production and diversification of key classes impractical. One pertinent example is the glycopeptide antibiotics (GPAs), which are highly challenging to synthesize due to their heavily cross-linked structures. Here, we report an optimized method that generates >75% tricyclic peptides from synthetic precursors in order to explore the acceptance of novel GPA precursor peptides by these key existent biosynthetic enzymes.

Kistamicin biosynthesis reveals the biosynthetic requirements for production of highly crosslinked glycopeptide antibiotics.

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster.

Studying trans-acting enzymes that target carrier protein-bound amino acids during nonribosomal peptide synthesis.

Nonribosomal peptide biosynthesis is a complex enzymatic assembly responsible for producing a great diversity of bioactive peptide natural products. Due to the recurring arrangement of catalytic domains within these machineries, great interest has been shown in reengineering these pathways to produce novel, designer peptide products. However, in order to realize such ambitions, it is first necessary to develop a comprehensive understanding of the selectivity, mechanisms, and structure of these complex enzymes, which in turn requires significant in vitro experiments.

Investigating Cytochrome P450 specificity during glycopeptide antibiotic biosynthesis through a homologue hybridization approach.

Cytochrome P450 enzymes perform an impressive range of oxidation reactions against diverse substrate scaffolds whilst generally maintaining a conserved tertiary structure and active site chemistry. Within secondary metabolism, P450 enzymes play widespread and important roles in performing crucial modifications of precursor molecules, with one example of the importance of such reactions being found in the biosynthesis of the glycopeptide antibiotics (GPAs). In GPA biosynthesis P450s, known as Oxy enzymes, are key players in the cyclization of the linear GPA peptide precursor, which is a process that is both essential for their antibiotic activity and is the source of the synthetic challenge of these important antibiotics.

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism.

Covering: 2000 up to 2018 The cytochromes P450 (P450s) are a superfamily of heme-containing monooxygenases that perform diverse catalytic roles in many species, including bacteria. The P450 superfamily is widely known for the hydroxylation of unactivated C-H bonds, but the diversity of reactions that P450s can perform vastly exceeds this undoubtedly impressive chemical transformation. Within bacteria, P450s play important roles in many biosynthetic and biodegradative processes that span a wide range of secondary metabolite pathways and present diverse chemical transformations.

A route to diastereomerically pure phenylglycine thioester peptides: crucial intermediates for investigating glycopeptide antibiotic biosynthesis.

Non-ribosomal peptides contain an array of amino acid building blocks that can present challenges for the synthesis of important intermediates. Here, we report the synthesis of glycopeptide antibiotic (GPA) thioester peptides that retains the crucial stereochemical purity of the terminal phenylglycine residue, which we show is essential for the enzymatic GPA cyclisation cascade.

Wide Distribution of Foxicin Biosynthetic Gene Clusters in Strains - An Unusual Secondary Metabolite with Various Properties.

Tü6028 is known to produce the polyketide antibiotic polyketomycin. The deletion of the oxygenase gene led to a non-polyketomycin-producing mutant. Instead, novel compounds were produced by the mutant, which have not been detected before in the wild type strain.

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028.

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC.

The Draft Genome Sequence of Actinokineospora bangkokensis 44EHW Reveals the Biosynthetic Pathway of the Antifungal Thailandin Compounds with Unusual Butylmalonyl-CoA Extender Units.

We report the draft genome sequence of 44EHW, the producer of the antifungal polyene compounds, thailandins A and B. The sequence contains 7.45 Mb, 74.

Thailandins A and B, New Polyene Macrolactone Compounds Isolated from Actinokineospora bangkokensis Strain 44EHW(T), Possessing Antifungal Activity against Anthracnose Fungi and Pathogenic Yeasts.

Two new polyene macrolactone antibiotics, thailandins A, 1, and B, 2, were isolated from the fermentation broth of rhizosphere soil-associated Actinokineospora bangkokensis strain 44EHW(T). The new compounds from this strain were purified using semipreparative HPLC and Sephadex LH-20 gel filtration while following an antifungal activity guided fractionation. Their structures were elucidated through spectroscopic techniques including UV, HR-ESI-MS, and NMR.

The AT₂ domain of KirCI loads malonyl extender units to the ACPs of the kirromycin PKS.

The antibiotic kirromycin is assembled by a hybrid modular polyketide synthases (PKSs)/nonribosomal peptide synthetases (NRPSs). Five of six PKSs of this complex assembly line do not have acyltransferase (AT) and have to recruit this activity from discrete AT enzymes. Here, we show that KirCI is a discrete AT which is involved in kirromycin production and displays a rarely found three-domain architecture (AT₁-AT₂-ER).