Bacterial Pathogen Channels Medium-Sized Fatty Acids into Malleicyprol Biosynthesis.

Bacterial pathogens of the Burkholderia pseudomallei (BP) group cause life-threatening infections in both humans and animals. Critical for the virulence of these often antibiotic-resistant pathogens is the polyketide hybrid metabolite malleicyprol, which features two chains, a short cyclopropanol-substituted chain and a long hydrophobic alkyl chain. The biosynthetic origin of the latter has remained unknown. Here, we report the discovery of novel overlooked malleicyprol congeners with varied chain lengths and identify medium-sized fatty acids as polyketide synthase (PKS) starter units that constitute the hydrophobic carbon tails. Mutational and biochemical analyses show that a designated coenzyme A-independent fatty acyl-adenylate ligase (FAAL, BurM) is essential for recruiting and activating fatty acids in malleicyprol biosynthesis. In vitro reconstitution of the BurM-catalyzed PKS priming reaction and analysis of ACP-bound building blocks reveal a key role of BurM in the toxin assembly. Insights into the function and role of BurM hold promise for the development of enzyme inhibitors as novel antivirulence therapeutics to combat infections with BP pathogens.

Pathogenic bacteria remodel central metabolic enzyme to build a cyclopropanol warhead.

Bacteria of the Burkholderia pseudomallei (BP) group pose a global health threat, causing the infectious diseases melioidosis, a common cause of pneumonia and sepsis, and glanders, a contagious zoonosis. A trait of BP bacteria is a conserved gene cluster coding for the biosynthesis of polyketides (malleicyprols) with a reactive cyclopropanol unit that is critical for virulence. Enzymes building this warhead represent ideal targets for antivirulence strategies but the biochemical basis of cyclopropanol formation is unknown. Here we describe the formation of the malleicyprol warhead. We show that BurG, an unusual NAD+-dependent member of the ketol-acid reductoisomerase family, constructs the strained cyclopropanol ring. Biochemical assays and a suite of eight crystal structures of native and mutated BurG with bound analogues and inhibitors provide snapshots of each step of the complex reaction mechanism, involving a concealed oxidoreduction and a C-S bond cleavage. Our findings illustrate a remarkable case of neofunctionalisation, where a biocatalyst from central metabolism has been evolutionarily repurposed for warhead production in pathogens.

Sequential Allylic Alcohol Formation by a Multifunctional Cytochrome†P450 Monooxygenase with Rare Redox Partners.

Caryoynencin is a toxic and antifungal fatty acid derivative produced by a number of plant-pathogenic and insect-protective bacteria (Trinickia caryophylli and Burkholderia spp.). In addition to the reactive tetrayne unit, the presence of an allylic alcohol moiety is critical for antimicrobial activities. By a combination of mutational analyses, heterologous expression and in vitro reconstitution experiments we show that the cytochrome P450 monooxygenase CayG catalyzes the complex transformation of a saturated carbon backbone into an allylic alcohol. Unexpectedly, CayG employs a ferritin-like protein (CayK) or a rubredoxin (CayL) component for electron transport. A desaturation-hydroxylation sequence was deduced from a time-course study and in vitro biotransformations with pathway intermediates, substrate analogues, protegencin congeners from Pseudomonas protegens Pf-5, and synthetic derivatives. This unusual multifunctional oxygenase may inspire future biocatalytic applications.

Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor.

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET-domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS-PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli.

Biosynthesis of Diverse Antimicrobial and Antiproliferative Acyloins in Anaerobic Bacteria.

Metabolic profiling and genome mining revealed that anaerobic bacteria have the potential to produce acyloin natural products. In addition to sattazolin A and B, three new sattazolin congeners and a novel acyloin named clostrocyloin were isolated from three strains of Clostridium beijerinckii, a bacterium used for industrial solvent production. Bioactivity profiling showed that the sattazolin derivatives possess antimicrobial activities against mycobacteria and pseudomonads with only low cytotoxicity. Clostrocyloin was found to be mainly active against fungi. The thiamine diphosphate (ThDP)-dependent sattazolin-producing synthase was identified in silico and characterized both in vivo and in in vitro enzyme assays. A related acyloin synthase from the clostrocyloin producer was shown to be responsible for the production of the acyloin core of clostrocyloin. The biotransformation experiments provided first insights into the substrate scope of the clostrocyloin synthase and revealed biosynthetic intermediates.

Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification.

Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

Regioselective Dichlorination of a Non-Activated Aliphatic Carbon Atom and Phenolic Bismethylation by a Multifunctional Fungal Flavoenzyme.

The regioselective functionalization of non-activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono- and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O-methylation. AoiQ was successfully reconstituted in vivo and in vitro, unequivocally showing that this FADH2 -dependent enzyme is uniquely capable of the stepwise gem-dichlorination of a non-activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi.