Intermediates Released from a Polyether-Producing Polyketide Synthase Provide Insight into the Mechanism of Oxidative Cyclization

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5′-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C–C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A−I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature’s arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.

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Structural and evolutionary relationships of “AT-less” type I polyketide synthase ketosynthases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515460112 ◽

2015 ◽

Vol 112 (41) ◽

pp. 12693-12698 ◽

Cited By ~ 37

Author(s):

Jeremy R. Lohman ◽

Ming Ma ◽

Jerzy Osipiuk ◽

Boguslaw Nocek ◽

Youngchang Kim ◽

...

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Polyketide Synthase ◽

Acyl Carrier Protein ◽

Structural Diversity ◽

Type I ◽

Substrate Specificities ◽

In Trans ◽

Canonical Type ◽

Insight Into

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.

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Docking analysis of hexanoic acid and quercetin with seven domains of polyketide synthase A provided insight into quercetin-mediated aflatoxin biosynthesis inhibition in Aspergillus flavus

3 Biotech ◽

10.1007/s13205-019-1675-y ◽

2019 ◽

Vol 9 (4) ◽

Cited By ~ 3

Author(s):

Shraddha Tiwari ◽

Sonia K. Shishodia ◽

Jata Shankar

Keyword(s):

Aspergillus Flavus ◽

Polyketide Synthase ◽

Hexanoic Acid ◽

Aflatoxin Biosynthesis ◽

Docking Analysis ◽

Insight Into

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Structural and Data Science-Driven Analysis to Assess Substrate Specificity of Diketopiperazine Reverse Prenyltransferase NotF: Cascade Biocatalytic Synthesis of (–)-Eurotiumin A

10.33774/chemrxiv-2021-gmv5j ◽

2021 ◽

Author(s):

Samantha P. Kelly ◽

Vikram V. Shende ◽

Autumn R. Flynn ◽

Qingyun Dan ◽

Ying Ye ◽

...

Keyword(s):

Data Science ◽

Early Stage ◽

Oxidative Cyclization ◽

Biologically Active ◽

Full Potential ◽

One Pot ◽

Unique Approach ◽

Substrate Promiscuity ◽

Insight Into

Prenyltransfer is an early-stage carbon–hydrogen bond (C–H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Towards the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). In the biosynthesis of cytotoxic notoamide metabolites, PT NotF is responsible for catalyzing the first tailoring event of C2 reverse prenyltransfer of brevianamide F (cyclo(L-Trp-L-Pro)). Obtaining a high-resolution crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent exposed substrate binding site, intimating NotF may possess significant substrate promiscuity. To assess the full potential of NotF’s broad substrate selectivity, we synthesized a panel of 30 tryptophanyl DKPs with a suite of sterically and electronically differentiated amino acids, which were selectively prenylated by NotF in often synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library enabled the development of a descriptive statistical model that provided insight into the origins of NotF’s substrate promiscuity. Through this unique approach for understanding enzyme scope, we identified key substrate descriptors such as electrophilicity, size, and flexibility, that govern enzymatic turnover by NotF. Additionally, we demonstrated the ability to couple NotF-catalyzed prenyltransfer with oxidative cyclization using recently characterized flavin monooxygenase, BvnB, from the brevianamide biosynthetic pathway. This one-pot, in vitro biocatalytic cascade proceeds with exceptional substrate recognition, and enabled the first chemoenzymatic synthesis of the marine fungal natural product, (–)-eurotiumin A, in three steps and 60% overall yield.

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Biosynthetic Gene Cluster for the Polyenoyltetramic Acid α-Lipomycin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00007-06 ◽

2006 ◽

Vol 50 (6) ◽

pp. 2113-2121 ◽

Cited By ~ 54

Author(s):

C. Bihlmaier ◽

E. Welle ◽

C. Hofmann ◽

K. Welzel ◽

A. Vente ◽

...

Keyword(s):

Gene Cluster ◽

Polyketide Synthase ◽

Biosynthetic Gene Cluster ◽

Open Reading Frames ◽

Biosynthetic Gene ◽

Peptide Synthetases ◽

Insight Into ◽

Reading Frames ◽

Module System

ABSTRACT The gram-positive bacterium Streptomyces aureofaciens Tü117 produces the acyclic polyene antibiotic α-lipomycin. The entire biosynthetic gene cluster (lip gene cluster) was cloned and characterized. DNA sequence analysis of a 74-kb region revealed the presence of 28 complete open reading frames (ORFs), 22 of them belonging to the biosynthetic gene cluster. Central to the cluster is a polyketide synthase locus that encodes an eight-module system comprised of four multifunctional proteins. In addition, one ORF shows homology to those for nonribosomal peptide synthetases, indicating that α-lipomycin belongs to the classification of hybrid peptide-polyketide natural products. Furthermore, the lip cluster includes genes responsible for the formation and attachment of d-digitoxose as well as ORFs that resemble those for putative regulatory and export functions. We generated biosynthetic mutants by insertional gene inactivation. By analysis of culture extracts of these mutants, we could prove that, indeed, the genes involved in the biosynthesis of lipomycin had been cloned, and additionally we gained insight into an unusual biosynthesis pathway.

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New Insight into Antimicrobial Compounds from Food and Marine-Sourced Carnobacterium Species through Phenotype and Genome Analyses

Microorganisms ◽

10.3390/microorganisms8071093 ◽

2020 ◽

Vol 8 (7) ◽

pp. 1093

Author(s):

Simon Begrem ◽

Flora Ivaniuk ◽

Frédérique Gigout-Chevalier ◽

Laetitia Kolypczuk ◽

Sandrine Bonnetot ◽

...

Keyword(s):

Polyketide Synthase ◽

Biosynthetic Gene Cluster ◽

Ecological Niches ◽

Antimicrobial Compounds ◽

Biosynthetic Gene ◽

Peptide Synthetase ◽

Seafood Products ◽

Genome Analyses ◽

Encoding Genes ◽

Insight Into

Carnobacterium maltaromaticum and Carnobacterium divergens, isolated from food products, are lactic acid bacteria known to produce active and efficient bacteriocins. Other species, particularly those originating from marine sources, are less studied. The aim of the study is to select promising strains with antimicrobial potential by combining genomic and phenotypic approaches on large datasets comprising 12 Carnobacterium species. The biosynthetic gene cluster (BGCs) diversity of 39 publicly available Carnobacterium spp. genomes revealed 67 BGCs, distributed according to the species and ecological niches. From zero to six BGCs were predicted per strain and classified into four classes: terpene, NRPS (non-ribosomal peptide synthetase), NRPS-PKS (hybrid non-ribosomal peptide synthetase-polyketide synthase), RiPP (ribosomally synthesized and post-translationally modified peptide). In parallel, the antimicrobial activity of 260 strains from seafood products was evaluated. Among the 60% of active strains, three genomes were sequenced and submitted to a dereplication process. C. inhibens MIP2551 produced a high amountof H2O2, probably thanks to the presence of four oxidase-encoding genes. C. maltaromaticum EBP3019 and SF668 strains were highly efficient against Listeria monocytogenes. A new extracellular 16 kDa unmodified bacteriocin in the EBP3019 strain and five different bacteriocins in SF668 were highlighted. In this study, the overview of antimicrobial BGC and inhibitory activities of Carnobacterium spp. allowed the prediction of potential innovative natural products that could be relevant for biotechnological applications.

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