Abstract Rifamycin-derived drugs, including rifampin, rifabutin, rifapentine, and rifaximin, have long been used as first-line therapies for the treatment of tuberculosis and other deadly infections. However, the late steps leading to the biosynthesis of the industrially important rifamycin SV and B remain largely unknown. Here, we characterize a network of reactions underlying the biosynthesis of rifamycin SV, S, L, O, and B. The two-subunit transketolase Rif15 and the cytochrome P450 enzyme Rif16 are found to mediate, respectively, a unique C–O bond formation in rifamycin L and an atypical P450 ester-to-ether transformation from rifamycin L to B. Both reactions showcase interesting chemistries for these two widespread and well-studied enzyme families.
<p>Fungal bicyclo[2.2.2]diazaoctane indole alkaloids demonstrate intriguing structures and a wide spectrum of biological activities. Although biomimetic total syntheses have been completed for representative compounds of this structural family, the details of their biogenesis have remained largely uncharacterized. Among them, Brevianamide A represents the most basic form within this class bearing a dioxopiperazine core structure and a rare 3-<i>spiro</i>-<i>psi</i>-indoxyl skeleton. Here, we identified the Brevianamide A biosynthetic gene cluster from <i>Penicillium brevicompacticum</i> NRRL 864 and fully elucidated the metabolic pathway by targeted gene disruption, heterologous expression, precursor incorporation studies, and <i>in vitro</i> biochemical analysis. In particular, we determined that BvnE is a cofactor-independent isomerase that is essential for selective production of Brevianamide A. Based on a high resolution crystal structure of BvnE, molecular modeling, mutational analysis, and computational studies provided new mechanistic insights into the diastereoselective formation of the 3-<i>spiro</i>-<i>psi</i>-indoxyl moiety in Brevianamide A. This occurs through a biocatalyst controlled semi-Pinacol rearrangement and a subsequent spontaneous intramolecular [4+2] <i>hetero</i>-Diels-Alder cycloaddition.</p>
A cryptic terpenoid biosynthetic gene cluster ven was characterized from the model actinomycetic strain Streptomyces venezuelae ATCC 15439 through a genome mining approach. Silent gene activation and heterologous expression of ven led to the discovery of two diterpene metabolites venezuelaene A (1) and B (2, 5-oxo-venezuelaene A), both of which bear a 5–5–6–7 tetracyclic skeleton and show fragrance. A class I diterpene synthase VenA with an atypical 115DSFVSD120 motif was revealed to catalyze the cyclization of geranylgeranyl pyrophosphate (GGPP) generated by the GGPP synthase VenD, giving rise to 1. The unique cyclization mechanism was elucidated by 13C-tracer NMR experiments. The oxidative transformation of 1 into 2 was also characterized to be mediated by the cytochrome P450 enzyme VenC.
Bafilomycin A1 is the representative compound of the plecomacrolide natural product family. This 16-membered ring plecomacrolide has potent antifungal and vacuolar H+-ATPase inhibitory activities. In our previous work, we identified a bafilomycin biosynthetic gene cluster (baf) from the marine bacterium Streptomyces lohii ATCC BAA-1276, wherein a luxR family regulatory gene orf1 and an afsR family regulatory gene bafG were revealed based on bioinformatics analysis. In this study, the positive regulatory roles of orf1 and bafG for bafilomycin biosynthesis are characterized through gene inactivation and overexpression. Compared to the wild-type S. lohii strain, the knockout of either orf1 or bafG completely abolished the production of bafilomycins. The overexpression of orf1 or bafG led to 1.3- and 0.5-fold increased production of bafilomycins, respectively. A genetically engineered S. lohii strain (SLO-08) with orf1 overexpression and inactivation of the biosynthetic genes orf2 and orf3, solely produced bafilomycin A1 with the titer of 535.1 ± 25.0 mg/L in an optimized fermentation medium in shaking flasks. This recombinant strain holds considerable application potential in large-scale production of bafilomycin A1 for new drug development.
The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN+ protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.
Friedel–Crafts acylation (FCA) is a highly beneficial approach in organic chemistry for creating the important C–C bonds that are necessary for building intricate frameworks between aromatic substrates and an acyl group. However, there are few reports about enzyme catalyzed FCA reactions. In this study, 4-acyl-5-aminoimidazole alkaloids (AAIAs), streptimidazoles A–C (1–3), and the enantiopure (+)-nocarimidazole C (4) as well as their ribosides, streptimidazolesides A–D (5–8), were identified from the fermentation broth of Streptomyces sp. OUCMDZ-944 or heterologous S. coelicolor M1154 mutant. The biosynthetic gene cluster (smz) was identified, and the biosynthetic pathway of AAIAs was elucidated for the first time. In vivo and in vitro studies proved the catalytic activity of the four essential genes smzB, -C, -E, and -F for AAIAs biosynthesis and clarified the biosynthetic process of the alkaloids. The ligase SmzE activates fatty acyl groups and connects them to the acyl carrier protein (ACP) holo-SmzF. Then, the acyl group is transferred onto the key residue Cys49 of SmzB, a new Friedel–Crafts acyltransferase (FCase). Subsequently, the FCA reaction between the acyl groups and 5-aminoimidazole ribonucleotide (AIR) occurs to generate the key intermediate AAIA-nucleotides catalyzed by SmzB. Finally, the hydrolase SmzC catalyzes the N-glycosidic bond cleavage of the intermediates to form AAIAs. Structural simulation, molecular modeling, and mutational analysis of SmzB showed that Tyr26, Cys49, and Tyr93 are the key catalytic residues in the C–C bond formation of the acyl chain of AAIAs, providing mechanistic insights into the enzymatic FCA reaction.
Significance Here, we elucidate the full biosynthetic pathway of the fungal natural product mycophenolic acid (MPA). Besides the intriguing enzymatic mechanisms, we reveal that the MPA biosynthetic enzymes are elegantly compartmentalized; the oxygenase MpaB′ is the long-sought enzyme responsible for initiating the oxidative cleavage of the farnesyl side chain; and the subcellular localization of the acyl-coenzyme A hydrolase MpaH′ in peroxisomes is required for the unique cooperation between biosynthetic and β-oxidation catabolism machineries. This work highlights the importance of a cell biology perspective for understanding the underexplored organelle-associated essential catalytic mechanisms in natural product biosynthesis of fungi and other higher organisms. The insights gained in our study will benefit future efforts for both industrial strain improvement and novel drug development.
Cytochrome P450 enzymes are highly diversified biocatalysts associated with steroid biosynthesis, xenobiotic metabolism, biosynthesis of natural products, and industrial oxidation reactions. A typical P450 catalytic cycle requires sequential transfer of two electrons from NAD(P)H to the heme-iron reactive center for O2 activation. For the most abundant bacterial Class I P450 systems, this important process is usually mediated by two redox partner proteins including an FAD-containing ferredoxin reductase (FdR) and a small iron–sulfur protein, ferredoxin (Fdx). However, it is often unclear which pair of Fdx and FdR among multiple redox partners is the optimal one for a specific Class I P450 enzyme. To address this important but underexplored question, herein, a reaction matrix network with 16 Fdxs, 8 FdRs, and 6 P450s (against 7 substrates) was constituted. By analyzing the reactivity profiles of 896 P450 reactions, together with phylogenetic analysis, redox potential measurements, structural simulations, and Fdx-P450 molecular docking, we provide important mechanistic insights into the recognition and interactions between bacterial Class I P450 enzymes and redox partners.
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