Absolute Configuration of 4-Chloroisoleucine in Cyclolithistide A: An Antifungal Peptide Lactone Discovered in Marine Lithistid Sponges.

Cyclolithistide A is a peptide lactone isolated from marine lithistid sponges. Its entire structure, including absolute configurations, has been reported except the relative and absolute configurations of its characteristic residue, 4-chloroisoleucine (4-CIle). We synthesized four isomers of 4-CIle from furfural-derived N-Boc imine and propionaldehyde. Analysis of the acid hydrolysate of cyclolithistide A and the synthetic samples of 4-CIle after derivatization with l- and d-FDAA permitted us to propose the absolute configuration of the 4-chloroisoleucine residue in cyclolithistide A as 2S,3R,4R.

A Secreted BBE-Like Enzyme Acting as a Drug-Binding Efflux Carrier Confers Microbial Self-Resistance to Mitomycin C.

The berberine bridge enzyme (BBE)-like flavoproteins have attracted continuous attention for their capability to catalyze various oxidative reactions. Here we demonstrate that MitR, a secreted BBE-like enzyme, functions as a special drug-binding efflux protein evolved from quinone reductase. Moreover, this protein provides self-resistance to its hosts toward the DNA-alkylating agent mitomycin C with a distinctive strategy, featured by independently performing drug binding and efflux.

Identification and characterization of a strong constitutive promoter stnYp for activating biosynthetic genes and producing natural products in streptomyces.

BACKGROUND: Streptomyces are well known for their potential to produce various pharmaceutically active compounds, the commercial development of which is often limited by the low productivity and purity of the desired compounds expressed by natural producers. Well-characterized promoters are crucial for driving the expression of target genes and improving the production of metabolites of interest. RESULTS: A strong constitutive promoter, stnYp, was identified in Streptomyces flocculus CGMCC4.1223 and was characterized by its effective activation of silent biosynthetic genes and high efficiency of heterologous gene expression. The promoter stnYp showed the highest activity in model strains of four Streptomyces species compared with the three frequently used constitutive promoters ermEp*, kasOp*, and SP44. The promoter stnYp could efficiently activate the indigoidine biosynthetic gene cluster in S. albus J1074, which is thought to be silent under routine laboratory conditions. Moreover, stnYp was found suitable for heterologous gene expression in different Streptomyces hosts. Compared with the promoters ermEp*, kasOp*, and SP44, stnYp conferred the highest production level of diverse metabolites in various heterologous hosts, including the agricultural-bactericide aureonuclemycin and the antitumor compound YM-216391, with an approximately 1.4 - 11.6-fold enhancement of the yields. Furthermore, the purity of tylosin A was greatly improved by overexpressing rate-limiting genes through stnYp in the industrial strain. Further, the yield of tylosin A was significantly elevated to 10.30 ± 0.12 g/L, approximately 1.7-fold higher than that of the original strain. CONCLUSIONS: The promoter stnYp is a reliable, well-defined promoter with strong activity and broad suitability. The findings of this study can expand promoter diversity, facilitate genetic manipulation, and promote metabolic engineering in multiple Streptomyces species.

Trioxacarcin A Interactions with G-Quadruplex DNA Reveal Its Potential New Targets as an Anticancer Agent.

Trioxacarcin (TXN) A was reported to be an anticancer agent through alkylation of dsDNA. G-quadruplex DNA (G4-DNA) is frequently formed in the promoter regions of oncogenes and the ends of telomerase genes, considered as promising drug targets for anticancer therapy. There are no reports about TXN A interactions with G4-DNA. Here, we tested TXN A's interactions with several G4-DNA oligos with parallel, antiparallel, or hybrid folding, respectively. We demonstrated that TXN A preferred to alkylate one flexible guanine in the loops of parallel G4-DNA. The position of the alkylated guanine is in favor of interactions of G4-DNA with TXN A. The structure of TXN A covalently bound RET G4-DNA indicated that TXN A alkylation on RET G4-DNA stabilizes the G4-DNA conformation. These studies opened a new window of how TXN A interacted with G4-DNA, which might hint a new mode of its function as an anticancer agent.

Dihydrofolate reductase-like protein inactivates hemiaminal pharmacophore for self-resistance in safracin biosynthesis.

Dihydrofolate reductase (DHFR), a housekeeping enzyme in primary metabolism, has been extensively studied as a model of acid-base catalysis and a clinic drug target. Herein, we investigated the enzymology of a DHFR-like protein SacH in safracin (SAC) biosynthesis, which reductively inactivates hemiaminal pharmacophore-containing biosynthetic intermediates and antibiotics for self-resistance. Furthermore, based on the crystal structure of SacH-NADPH-SAC-A ternary complexes and mutagenesis, we proposed a catalytic mechanism that is distinct from the previously characterized short-chain dehydrogenases/reductases-mediated inactivation of hemiaminal pharmacophore. These findings expand the functions of DHFR family proteins, reveal that the common reaction can be catalyzed by distinct family of enzymes, and imply the possibility for the discovery of novel antibiotics with hemiaminal pharmacophore.

Biosynthesis of DNA-Alkylating Antitumor Natural Products.

DNA-alkylating natural products play an important role in drug development due to their significant antitumor activities. They usually show high affinity with DNA through different mechanisms with the aid of their unique scaffold and highly active functional groups. Therefore, the biosynthesis of these natural products has been extensively studied, especially the construction of their pharmacophores. Meanwhile, their producing strains have evolved corresponding self-resistance strategies to protect themselves. To further promote the functional characterization of their biosynthetic pathways and lay the foundation for the discovery and rational design of DNA alkylating agents, we summarize herein the progress of research into DNA-alkylating antitumor natural products, including their biosynthesis, modes of action, and auto-resistance mechanisms.

Discovery of glycosylated naphthacemycins and elucidation of the glycosylation.

Two glycosylated naphthacemycins (naphthacemycins D1 and D2) were identified in Streptomyces sp. N12W1565. These two compounds not only showed antimicrobial potential against bacteria but also exhibited more aqueous solubility than naphthacemycins. Furthermore, the whole genome of Streptomyces sp. N12W1565 has been sequenced, the natY gene, located outside the biosynthetic gene cluster encoding a D-glucose glycosyltransferase, was identified to mediate glycosylation in the phenolic hydroxyl of the naphthacemycin core scaffold. Glycosyltransferase was elucidated in vitro by using a homologous enzyme, which showed potential as a biocatalyst.

Base excision repair system targeting DNA adducts of trioxacarcin/LL-D49194 antibiotics for self-resistance.

Two families of DNA glycosylases (YtkR2/AlkD, AlkZ/YcaQ) have been found to remove bulky and crosslinking DNA adducts produced by bacterial natural products. Whether DNA glycosylases eliminate other types of damage formed by structurally diverse antibiotics is unknown. Here, we identify four DNA glycosylases-TxnU2, TxnU4, LldU1 and LldU5-important for biosynthesis of the aromatic polyketide antibiotics trioxacarcin A (TXNA) and LL-D49194 (LLD), and show that the enzymes provide self-resistance to the producing strains by excising the intercalated guanine adducts of TXNA and LLD. These enzymes are highly specific for TXNA/LLD-DNA lesions and have no activity toward other, less stable alkylguanines as previously described for YtkR2/AlkD and AlkZ/YcaQ. Similarly, TXNA-DNA adducts are not excised by other alkylpurine DNA glycosylases. TxnU4 and LldU1 possess unique active site motifs that provide an explanation for their tight substrate specificity. Moreover, we show that abasic (AP) sites generated from TxnU4 excision of intercalated TXNA-DNA adducts are incised by AP endonuclease less efficiently than those formed by 7mG excision. This work characterizes a distinct class of DNA glycosylase acting on intercalated DNA adducts and furthers our understanding of specific DNA repair self-resistance activities within antibiotic producers of structurally diverse, highly functionalized DNA damaging agents.

Heterologous characterization of mechercharmycin A biosynthesis reveals alternative insights into post-translational modifications for RiPPs.

Mechercharmycin A (MCM-A) is a marine natural product belonging to a family of polyazole cyclopeptides with remarkable bioactivities and unique structures. Identification, heterologous expression, and genetic characterizations of the MCM biosynthetic gene cluster in Bacillus subtilis revealed that it is a ribosomally synthesized and post-translationally modified peptide (RiPP) possessing complex with distinctive modifications. Based on this heterologous expression system, two MCM analogs with comparable antitumor activity are generated by engineering the biosynthetic pathway. Combinatorial co-production of a precursor peptide with different modifying enzymes in Escherichia coli identifies a different timing of modifications, showing that a tRNAGlu-dependent highly regioselective dehydration is the first modification step, followed by polyazole formation through heterocyclization and dehydrogenation in an N- to C-terminal direction. Therefore, a rational biosynthetic pathway of MCMs is proposed, which unveils a subfamily of azol(in)e-containing RiPPs and sets the stage for further investigations of the enzymatic mechanism and synthetic biology.

A Cryptic Palmitoyl Chain Involved in Safracin Biosynthesis Facilitates Post-NRPS Modifications.

This study confirmed the participation of a cryptic palmitoyl fatty acyl chain in the biosynthesis of safracin and unraveled a previously ignored peptidase for the removal of the precursor. Furthermore, the post-assembly line tailoring steps are extensively studied in terms of the methyltransferase SacI-catalyzed N-methylation and the FAD-dependent monooxygenase SacJ-catalyzed A-ring oxidation. The timing of these post-NRPS steps is also addressed in this work.

Newly Discovered Mechanisms of Antibiotic Self-Resistance with Multiple Enzymes Acting at Different Locations and Stages.

Self-resistance determinants are essential for the biosynthesis of bioactive natural products and are closely related to drug resistance in clinical settings. The study of self-resistance mechanisms has long moved forward on the discovery of new resistance genes and the characterization of enzymatic reactions catalyzed by these proteins. However, as more examples of self-resistance have been reported, it has been revealed that the enzymatic reactions contribute to self-protection are not confined to the cellular location where the final toxic compounds are present. In this review, we summarize representative examples of self-resistance mechanisms for bioactive natural products functional at different cell locations to explore the models of resistance strategies involved. Moreover, we also highlight those resistance determinants that are widespread in nature and describe the applications of self-resistance genes in natural product mining to interrogate the landscape of self-resistance genes in drug resistance-related new drug discovery.

Reductive inactivation of the hemiaminal pharmacophore for resistance against tetrahydroisoquinoline antibiotics.

Antibiotic resistance is becoming one of the major crises, among which hydrolysis reaction is widely employed by bacteria to destroy the reactive pharmacophore. Correspondingly, antibiotic producer has canonically co-evolved this approach with the biosynthetic capability for self-resistance. Here we discover a self-defense strategy featuring with reductive inactivation of hemiaminal pharmacophore by short-chain dehydrogenases/reductases (SDRs) NapW and homW, which are integrated with the naphthyridinomycin biosynthetic pathway. We determine the crystal structure of NapW·NADPH complex and propose a catalytic mechanism by molecular dynamics simulation analysis. Additionally, a similar detoxification strategy is identified in the biosynthesis of saframycin A, another member of tetrahydroisoquinoline (THIQ) antibiotics. Remarkably, similar SDRs are widely spread in bacteria and able to inactive other THIQ members including the clinical anticancer drug, ET-743. These findings not only fill in the missing intracellular events of temporal-spatial shielding mode for cryptic self-resistance during THIQs biosynthesis, but also exhibit a sophisticated damage-control in secondary metabolism and general immunity toward this family of antibiotics.

Identification and Characterization of Enzymes Catalyzing Early Steps in Miharamycin and Amipurimycin Biosynthesis.

The biochemical elucidation of the early biosynthetic pathways of miharamycins and amipurimycin revealed the roles of several enzymes, which include GMP hydrolase, represented by MihD/ApmD, and hypothetical proteins, MihI/ApmI, unexpectedly exhibiting the dual function of the guanylglucuronic acid assembly and GMP cleavage. In addition, MihE, a carbonyl reductase that functions on the C2 branch of high-carbon sugars, and MihF, a rare guanine O-methyltransferase, were also functionally verified.

Structural evolution of a DNA repair self-resistance mechanism targeting genotoxic secondary metabolites.

Microbes produce a broad spectrum of antibiotic natural products, including many DNA-damaging genotoxins. Among the most potent of these are DNA alkylating agents in the spirocyclopropylcyclohexadienone (SCPCHD) family, which includes the duocarmycins, CC-1065, gilvusmycin, and yatakemycin. The yatakemycin biosynthesis cluster in Streptomyces sp. TP-A0356 contains an AlkD-related DNA glycosylase, YtkR2, that serves as a self-resistance mechanism against yatakemycin toxicity. We previously reported that AlkD, which is not present in an SCPCHD producer, provides only limited resistance against yatakemycin. We now show that YtkR2 and C10R5, a previously uncharacterized homolog found in the CC-1065 biosynthetic gene cluster of Streptomyces zelensis, confer far greater resistance against their respective SCPCHD natural products. We identify a structural basis for substrate specificity across gene clusters and show a correlation between in vivo resistance and in vitro enzymatic activity indicating that reduced product affinity-not enhanced substrate recognition-is the evolutionary outcome of selective pressure to provide self-resistance against yatakemycin and CC-1065.

A Flavin-Dependent Monooxygenase Mediates Divergent Oxidation of Rifamycin.

Rifamycins have been clinically utilized against mycobacterial infections for more than 50 years; however, their biosynthesis has not been fully elucidated. Here, on the basis of in vivo gene deletions, in vitro enzyme assays, isotope labeling, and site-directed mutations, we found that a flavin-dependent monooxygenase encoded by a rifamycin biosynthetic gene cluster, Rif-Orf17, not only converted the naphthoquinone chromophore of rifamycin S into benzo-γ-pyrone but also linearized rifamycin SV through phenolic hydroxylation. Both oxidation routes lead to inactivation of rifamycins.

Characterization of the Rifamycin-Degrading Monooxygenase From Rifamycin Producers Implicating Its Involvement in Saliniketal Biosynthesis.

Rifamycin derivatives, such as rifampicin, have potent antibiotic activity and have long been used in the clinic as mainstay components for the treatment of tuberculosis, leprosy, and AIDS-associated mycobacterial infections. However, the extensive usage of these antibiotics has resulted in the rapid development of bacterial resistance. The resistance mechanisms mainly include mutations of the rifamycin target RNA polymerase of bacteria and enzymatic modifications of rifamycin antibiotics. One modification is the recently characterized rifamycin degradation catalyzed by Rox enzymes, which belong to the widely occurring flavin monooxygenases. Intriguingly, our recent sequence analysis revealed the rifamycin producers also encode Rox homologs that are not yet characterized. In this work, we expanded the study of the Rox-catalyzed rifamycin degradation. We first showed that the Rox proteins from rifamycin producers have the enzymatic rifamycin SV-degrading activity. Then we used the structurally diverse rifamycin compounds rifampicin and 16-demethylrifamycin W to probe the substrate scope and found that they each have a slightly different substrate scope. Finally, we demonstrated that Rox proteins can also catalyze the transformation of 16-demethylsalinisporamycin to 16-demethylsaliniketal A. Since 16-demethylsalinisporamycin and 16-demethylsaliniketal A are the counterpart analogs of salinisporamycin and saliniketal A, our biochemical findings not only uncover a previously uncharacterized self-resistance mechanism in the rifamycin producers, but also bridge the gap between the biosynthesis of the potential antitumor compound saliniketal A.

Characterization of Miharamycin Biosynthesis Reveals a Hybrid NRPS-PKS to Synthesize High-Carbon Sugar from a Complex Nucleoside.

Miharamycins are peptidyl nucleoside antibiotics with a unique branched C9 pyranosyl amino acid core and a rare 2-aminopurine moiety. Inactivation of 19 genes in the biosynthetic gene cluster and identification of several unexpected intermediates suggest an alternative biosynthetic pathway, which is further supported by feeding experiments and in vitro characterization of an unusual adenylation domain recognizing a complex nucleoside derivative as the substrate. These results thereby provide an unprecedented biosynthetic route of high-carbon sugar catalyzed by atypical hybrid nonribosomal peptide synthetase-polyketide synthase.

Recent advances in HemN-like radical S-adenosyl-l-methionine enzyme-catalyzed reactions.

Covering: 2012 to 2019HemN-like radical S-adenosyl-l-methionine (SAM) enzymes have been recently disclosed to catalyze diverse chemically challenging reactions from primary to secondary metabolic pathways. In this highlight, we summarize the reaction examples catalyzed by HemN-like enzymes to date and the enzymatic mechanisms reported. From the recent mechanistic investigations, we reason that there is a shared initiating mechanism wherein a characteristic SAM methylene radical is proposed to abstract a hydrogen atom from an sp3 carbon or add onto an sp2 carbon center although variations occur thereafter from reaction to reaction, as well as providing a brief insight into some future prospects.

Directed Biosynthesis of Iso-aclacinomycins with Improved Anticancer Activity.

A four-enzyme catalyzed hydroxy regioisomerization of anthracycline was integrated into the biosynthetic pathway of aclacinomycin A (ALM-A), to generate a series of iso-ALMs via directed combinatorial biosynthesis combined with precursor-directed mutasynthesis. Most of the newly acquired iso-ALMs exhibit obviously (1-5-fold) improved antitumor activity. Therefore, we not only developed iso-ALMs with potential as clinical drugs but also demonstrated the utility of this tailoring tool for modification of anthracycline antibiotics in drug discovery and development.