Novel transformation products from glucosinolate-derived thioglucose and isothiocyanates formed during cooking.

Glucosinolates are plant secondary metabolites occurring in Brassicaceae plants. Upon tissue disruption, these compounds can be enzymatically hydrolyzed into isothiocyanates, which are very reactive and can react with nucleophiles during thermal processes such as boiling. Here, a novel type of glucosinolate degradation product was identified resulting from the reaction of thioglucose with the isothiocyanates sulforaphane or allyl isothiocyanate during aqueous heating. The two heterocyclic compounds 4-hydroxy-3-(4-(methylsulfinyl)butyl)thiazolidine-2-thione and 3-allyl-4-hydroxythiazolidine-2-thione were isolated and their structure elucidated by NMR spectroscopy and high-resolution mass spectrometry. Based on a set of chemical experiments, a reaction mechanism was proposed. Finally, the formation of the two 3-alk(en)yl-4-hydroxythiazolidine-2-thiones was quantified in boiled cabbage samples using a standard addition method in which 92 pmol/g and 19 pmol/g fresh weight of the sulforaphane and allyl isothiocyanate derivatives were found, respectively.

Transdermal Delivery of a Hydrogen Sulphide Donor, ADT-OH Using Aqueous Gel Formulations for the Treatment of Impaired Vascular Function: an Ex Vivo Study.

PURPOSE: Hydrogen sulphide (H2S) is an important signalling molecule involved in the regulation of several physiological and pathophysiological processes. The objective of this study was to investigate the feasibility of transdermal delivery of ADT-OH, a H2S donor, by investigating the transdermal flux of aqueous gels loaded with penetration enhancers or liposomes. Furthermore, we explored the ability of permeated ADT-OH to promote angiogenesis and mitochondrial bioenergetics in HUVEC cells. METHODS: Aqueous hypromellose gels (5% w/v) were prepared with up to 10% v/v propylene glycol (PG) or deformable liposomes with 0.025% w/w ADT-OH. ADT-OH permeation from formulations across excised murine skin into PBS was quantified over 24 h using HPLC-UV detection. Media was collected and applied to HUVEC cells to evidence ADT-OH functionality following permeation. Tube formation assays were performed as indicative of angiogenesis and mitochondrial oxygen consumption was evaluated using a Seahorse XF24. RESULTS: Increasing the loading of PG caused an increase in ADT-OH permeation rate across skin and a decrease in dermal drug retention whereas liposomal gels produced a slow-release profile. Treatment of HUVEC's using conditioned media collected from the ADT-OH loaded permeation studies enhanced tube formation and the basal oxygen consumption rates after 30 min of treatment. CONCLUSIONS: These findings demonstrate that transdermal delivery of ADT-OH may provide a promising approach in the treatment of impaired vascular function. Gels prepared with 10% v/v PG have the potential for use in conditions requiring rapid H2S release whereas liposomal loaded gels for treatment requiring sustained H2S release.

Thiocysteine lyases as polyketide synthase domains installing hydropersulfide into natural products and a hydropersulfide methyltransferase.

Nature forms S-S bonds by oxidizing two sulfhydryl groups, and no enzyme installing an intact hydropersulfide (-SSH) group into a natural product has been identified to date. The leinamycin (LNM) family of natural products features intact S-S bonds, and previously we reported an SH domain (LnmJ-SH) within the LNM hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line as a cysteine lyase that plays a role in sulfur incorporation. Here we report the characterization of an S-adenosyl methionine (SAM)-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin (GNM) biosynthesis, discovery of hydropersulfides as the nascent products of the GNM and LNM hybrid NRPS-PKS assembly lines, and revelation of three SH domains (GnmT-SH, LnmJ-SH, and WsmR-SH) within the GNM, LNM, and weishanmycin (WSM) hybrid NRPS-PKS assembly lines as thiocysteine lyases. Based on these findings, we propose a biosynthetic model for the LNM family of natural products, featuring thiocysteine lyases as PKS domains that directly install a -SSH group into the GNM, LNM, or WSM polyketide scaffold. Genome mining reveals that SH domains are widespread in Nature, extending beyond the LNM family of natural products. The SH domains could also be leveraged as biocatalysts to install an -SSH group into other biologically relevant scaffolds.

4-Alkyl-1,2,4-triazole-3-thione analogues as metallo-ß-lactamase inhibitors.

In Gram-negative bacteria, the major mechanism of resistance to ß-lactam antibiotics is the production of one or several ß-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-ß-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with Ki values in the µM to sub-µM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2.

Mitochondrial hydrogen sulfide supplementation improves health in the C. elegans Duchenne muscular dystrophy model.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle degeneration and weakness due to mutations in the dystrophin gene. The symptoms of DMD share similarities with those of accelerated aging. Recently, hydrogen sulfide (H2S) supplementation has been suggested to modulate the effects of age-related decline in muscle function, and metabolic H2S deficiencies have been implicated in affecting muscle mass in conditions such as phenylketonuria. We therefore evaluated the use of sodium GYY4137 (NaGYY), a H2S-releasing molecule, as a possible approach for DMD treatment. Using the dys-1(eg33) Caenorhabditis elegans DMD model, we found that NaGYY treatment (100 µM) improved movement, strength, gait, and muscle mitochondrial structure, similar to the gold-standard therapeutic treatment, prednisone (370 µM). The health improvements of either treatment required the action of the kinase JNK-1, the transcription factor SKN-1, and the NAD-dependent deacetylase SIR-2.1. The transcription factor DAF-16 was required for the health benefits of NaGYY treatment, but not prednisone treatment. AP39 (100 pM), a mitochondria-targeted H2S compound, also improved movement and strength in the dys-1(eg33) model, further implying that these improvements are mitochondria-based. Additionally, we found a decline in total sulfide and H2S-producing enzymes in dystrophin/utrophin knockout mice. Overall, our results suggest that H2S deficit may contribute to DMD pathology, and rectifying/overcoming the deficit with H2S delivery compounds has potential as a therapeutic approach to DMD treatment.

4-Amino-1,2,4-triazole-3-thione-derived Schiff bases as metallo-ß-lactamase inhibitors.

Resistance to ß-lactam antibiotics in Gram-negatives producing metallo-ß-lactamases (MBLs) represents a major medical threat and there is an extremely urgent need to develop clinically useful inhibitors. We previously reported the original binding mode of 5-substituted-4-amino/H-1,2,4-triazole-3-thione compounds in the catalytic site of an MBL. Moreover, we showed that, although moderately potent, they represented a promising basis for the development of broad-spectrum MBL inhibitors. Here, we synthesized and characterized a large number of 4-amino-1,2,4-triazole-3-thione-derived Schiff bases. Compared to the previous series, the presence of an aryl moiety at position 4 afforded an average 10-fold increase in potency. Among 90 synthetic compounds, more than half inhibited at least one of the six tested MBLs (L1, VIM-4, VIM-2, NDM-1, IMP-1, CphA) with Ki values in the µM to sub-µM range. Several were broad-spectrum inhibitors, also inhibiting the most clinically relevant VIM-2 and NDM-1. Active compounds generally contained halogenated, bicyclic aryl or phenolic moieties at position 5, and one substituent among o-benzoic, 2,4-dihydroxyphenyl, p-benzyloxyphenyl or 3-(m-benzoyl)-phenyl at position 4. The crystallographic structure of VIM-2 in complex with an inhibitor showed the expected binding between the triazole-thione moiety and the dinuclear centre and also revealed a network of interactions involving Phe61, Tyr67, Trp87 and the conserved Asn233. Microbiological analysis suggested that the potentiation activity of the compounds was limited by poor outer membrane penetration or efflux. This was supported by the ability of one compound to restore the susceptibility of an NDM-1-producing E. coli clinical strain toward several ß-lactams in the presence only of a sub-inhibitory concentration of colistin, a permeabilizing agent. Finally, some compounds were tested against the structurally similar di-zinc human glyoxalase II and found weaker inhibitors of the latter enzyme, thus showing a promising selectivity towards MBLs.

Discovery of Cryptic Largimycins in Streptomyces Reveals Novel Biosynthetic Avenues Enriching the Structural Diversity of the Leinamycin Family.

Largimycins are hybrid nonribosomal peptide-polyketides that constitute a new group of metabolites in the leinamycin family of natural products displaying unique structural features such as containing an oxazole instead of a thiazole ring or being oxime ester macrocycles, unprecedented in nature, rather than macrolactams. Their discovery in Streptomyces argillaceus and Streptomyces canus has relied on the activation of two homologous silent gene clusters by overexpressing a transcriptional activator and cultivating in specific media. The proposed biosynthesis of largimycins includes the key action of the oxidoreductase LrgO, responsible for the formation of the oxime group involved in macrocyclization, and two putative cryptic biosynthetic steps consisting of chlorination of l-Thr by the NRPS loading module and incorporation of an olefinic exomethylene group by LrgJ PKS. The discovery of largimycins uncovers novel biosynthetic avenues employed in nature to enrich the structural diversity of leinamycins and provides tools for combinatorial biosynthesis.

A One-pot Synthesis of Novel Derivatives of Oxadiazine-4-thione, and its Antibacterial Activity, and Molecular Modeling Studies.

BACKGROUND: The synthesis of a novel series of oxadiazine-4-thione biological molecules was executed through the incorporation of the ortho-, meta-, and para-benzoyl isocyanates to the tetrabromophthalimide nucleus. OBJECTIVES: A one-pot multicomponent methodology in a solvent-free microwave irradiation environment was employed to afford this series of oxadiazine-4-thione, deriving a comparison with the conventional method. Subsequently, the yielded derivatives were subjected to further biological assessment. MATERIALS AND METHODS: The acquired results denoted that the one-pot procedure, which delivered products in a 2-4 min. interval, was more efficient in evaluation against the classical method, which consumed a 1-2:30 hr. interval. RESULTS: The application of the antibacterial analyses was subjected to all the compounds, resulting in molecules 6a and 6c demonstrating the highest activity regarding Aspergillus Favus; molecules 5b and 5c exhibiting an equivalent level of activity towards E-coli and Fusarium Moniliform; and molecules 4b, 4c, 5b, and 5c presenting an identical level of activity to the aforementioned derivatives involving Staphylococcus. Concluison: Molecular modeling studies by the MOE, the preceding antibacterial behavior was conducted to advocate the newly prepared compounds. Moreover, the spectroscopic approaches were exploited to verify and establish the structures and mechanisms of the synthesized derivatives' reactions.

Type II non-ribosomal peptide synthetase proteins: structure, mechanism, and protein-protein interactions.

Covering: 1990 to 2019 Many medicinally-relevant compounds are derived from non-ribosomal peptide synthetase (NRPS) products. Type I NRPSs are organized into large modular complexes, while type II NRPS systems contain standalone or minimal domains that often encompass specialized tailoring enzymes that produce bioactive metabolites. Protein-protein interactions and communication between the type II biosynthetic machinery and various downstream pathways are critical for efficient metabolite production. Importantly, the architecture of type II NRPS proteins makes them ideal targets for combinatorial biosynthesis and metabolic engineering. Future investigations exploring the molecular basis or protein-protein recognition in type II NRPS pathways will guide these engineering efforts. In this review, we consolidate the broad range of NRPS systems containing type II proteins and focus on structural investigations, enzymatic mechanisms, and protein-protein interactions important to unraveling pathways that produce unique metabolites, including dehydrogenated prolines, substituted benzoic acids, substituted amino acids, and cyclopropanes.

Probing the high potency of pyrazolyl pyrimidinetriones and thioxopyrimidinediones as selective and efficient non-nucleotide inhibitors of recombinant human ectonucleotidases.

With the aim to discover novel, efficient and selective inhibitors of human alkaline phosphatase and nucleotide pyrophosphatase enzymes, two new series of pyrazolyl pyrimidinetriones (PPTs) (6a-g) and thioxopyrimidinediones (PTPs) (6h-n) were synthesized in good chemical yields using Knoevenagel condensation reaction between pyrazole carbaldehydes (4a-g) and pharmacologically active N-alkylated pyrimidinetrione (5a) and thioxopyrimidinedione (5b). The inhibition potential of the synthesized hybrid compounds was evaluated against human alkaline phosphatase (h-TNAP and h-IAP) and ectonucleotidase (h-NPP1 and h-NPP3) enzymes. Most of the tested analogs were highly potent with a variable degree of inhibition depending on the functionalized hybrid structure. The detailed structure-activity relationship (SAR) of PPT and PTP derivatives suggested that the compound with unsubstituted phenyl ring from PPT series led to selective and potent inhibition (6a; IC50 = 0.33 ±â€¯0.02 µM) of h-TNAP, whereas compound 6c selectively inhibited h-IAP isozyme with IC50 value of 0.86 ±â€¯0.04 µM. Similarly, compounds 6b and 6h were identified as the lead scaffolds against h-NPP1 and h-NPP3, respectively. The probable binding modes for the most potent inhibitors were elucidated through molecular docking analysis. Structure-activity relationships, mechanism of action, cytotoxic effects and druglikeness properties are also discussed.

Pyridoxal phosphate-dependent reactions in the biosynthesis of natural products.

Covering: up to mid-2018 Pyridoxal 5'-phosphate (PLP) is a versatile organic cofactor used to catalyze diverse reactions on amino acid, oxoacid, and amine substrates. Here we review the reactions catalyzed by PLP-dependent enzymes, highlighting enzymes reported in the natural product biosynthetic literature. We describe enzymes that catalyze transaminations, Claisen-like condensations, and ß- and γ-eliminations and substitutions, along with epimerizations, decarboxylations, and transaldolations. Finally, we describe a newly reported group of O2-, PLP-dependent enzymes. Altogether, natural product biosynthesis showcases the incredible versatility of PLP-dependent transformations for building chemical complexity.

Comparative evaluation of new dihydropyrimidine and dihydropyridine derivatives perturbing mitotic spindle formation.

AIM: The mitotic spindle plays a key role in cell division which makes it an important target in cancer therapy. In the present study the antiproliferative activity of 4-benzyl-5-phenyl-3,4-dihydropyrimidine-2(1H)-thione (1) and its pyridine bioisoster (2) were evaluated and compared with monastrol (MON), the first known cell-permeable small molecule which disrupts bipolar spindle formation by inhibiting Eg5-kinesin activity. RESULTS: Our data revealed that compound 2 showed higher antiproliferative activity than MON against MCF7 and A375 cell lines and comparable reversible cell cycle inhibition in G2/M phase. However, compound 2 produced distinct phenotype from monoastral spindles, and did not affect Eg5 ATPase activity. CONCLUSION: The activity of compound 2 may suggest its new promising anticancer mechanism (different than MON), targeting other component required for spindle bipolarity.

3H-1,2-dithiole-3-thione protects PC12 cells against amyloid beta 1-42 (Aß1-42) induced apoptosis via activation of the ERK1/2 pathway.

AIMS: Increasing evidence displays that deposition of aggregated ß-amyloid (Aß) leads to neuronal cell apoptosis, thus aggravates the pathological progression of Alzheimer's disease (AD). 3H-1,2-dithiole-3-thione (D3T) has been proved to exert neuroprotective effects. However, the effect of D3T on protecting against Aß-induced apoptosis and the underlying mechanism are unknown. MAIN METHODS: MTT, DCFH-DA assay, LDH release assay, Fluo-3 AM assay, Flow cytometry and Western blot were used to examine cell viability, ROS level, LDH release, intracellular Ca2+ concentration, cell apoptosis and related proteins level respectively. KEY FINDINGS: In the present study, we found that D3T pretreatment significantly increased cell viability and decreased reactive oxygen species (ROS) levels, lactate dehydrogenase (LDH) levels and the intracellular calcium concentration of rat pheochromocytoma (PC12) cells after Aß1-42 exposure. In addition, D3T pretreatment inhibited Aß1-42 induced cell apoptosis as well as protein levels of Bax and Caspase-3 in PC12 cells. Further, D3T markedly activated extracellular regulated protein kinase 1/2 (p-ERK1/2) but not PI3K/Akt signaling. Moreover, the protective effect of D3T against Aß1-42 induced apoptosis was abolished by the ERK1/2 pathway inhibitor PD98059 while PI3K inhibitor LY294002 had no significant effect. SIGNIFICANCE: Taken together, these findings suggest that D3T protects PC12 cells against Aß1-42 induced apoptosis through activation of the ERK1/2 pathway.

P450-Catalyzed Tailoring Steps in Leinamycin Biosynthesis Featuring Regio- and Stereoselective Hydroxylations and Substrate Promiscuities.

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown. Based on gene inactivation and chemical investigation of three mutant strains, we investigated the tailoring steps catalyzed by two cytochromes P450 (P450s), LnmA and LnmZ, in LNM biosynthesis. Our studies revealed that (i) LnmA and LnmZ regio- and stereoselectively hydroxylate the C-8 and C-4' positions, respectively, on the scaffold of LNM; (ii) both LnmA and LnmZ exhibit substrate promiscuity, resulting in multiple LNM analogs from several shunt pathways; and (iii) the C-8 and C-4' hydroxyl groups play important roles in the cytotoxicity of LNM analogs against different cancer cell lines, shedding light on the structure-activity relationships of the LNM scaffold and the LNM-type natural products in general. These studies set the stage for future biosynthetic pathway engineering and combinatorial biosynthesis of the LNM family of natural products for structure diversity and drug discovery.

Glucosinolate turnover in Brassicales species to an oxazolidin-2-one, formed via the 2-thione and without formation of thioamide.

Glucosinolates are found in plants of the order Brassicales and hydrolyzed to different breakdown products, particularly after tissue damage. In Barbarea vulgaris R.Br. (Brassicaceae), the dominant glucosinolate in the investigated "G-type" is glucobarbarin, (S)-2-hydroxy-2-phenylethylglucosinolate. Formation of the nitrile from glucobarbarin was observed in vitro, while a previously suggested thioamide (synonym thionamide) was not confirmed. Resedine (5-phenyl-1,3-oxazolidin-2-one) was detected after glucobarbarin hydrolysis in crushed B. vulgaris leaves and siliques, but not in intact parts. The abundance increased for several hours after completion of hydrolysis. The corresponding 1,3-oxazolidine-2-thione (OAT), with the common name barbarin, was also formed, and appeared to be the precursor of resedine. Addition of each of two non-endogenous OATs, (S)-5-ethyl-5-methylOAT and (R)-5-vinylOAT (R-goitrin), to a leaf homogenate resulted in formation of the corresponding 1,3-oxazolidin-2-ones (OAOs), confirming the metabolic connection of OAT to OAO. Formation of OAOs was inhibited by prior brief heating of the homogenate, suggesting enzyme involvement. We suggest the conversion of OATs to OAOs to be catalyzed by an enzyme ("oxazolidinethionase") responsible for turnover of OAT formed in intact plants. Resedine had been reported as an alkaloid from another species - Reseda luteola L. (Resedaceae) - naturally containing the glucosinolate glucobarbarin. However, resedine was not detected in intact R. luteola plants, but formed after tissue damage. The formation of resedine in two families suggests a broad distribution of putative OATases in the Brassicales; potentially involved in glucosinolate turnover that needs myrosinase activity as the committed step. In agreement with the proposed function of OATase, several candidate genes for myrosinases in glucosinolate turnover in intact plants were discovered in the B. vulgaris genome. We also suggest that biotechnological conversion of OATs to OAOs might improve the nutritional value of Brassicales protein. HPLC-MS/MS methods for detection of these glucobarbarin products are described.

Pharmacological activation of the Nrf2 pathway by 3H-1, 2-dithiole-3-thione is neuroprotective in a mouse model of Alzheimer disease.

Accumulating evidence suggests that oxidative stress induced by beta-amyloid (Aß) is implicated in the pathlogical progression of Alzheimer's disease (AD). 3H-1,2-dithiole-3-thione (D3T), the simplest compound of the sulfur-containing dithiolethiones, has been proved to be a strongly active antioxidant factor by regulation of the nuclear factor E2-related factor 2 (Nrf2). Previous study reported that D3T confers protection to AD cell model in vitro, however, the neuroprotective effect of D3T in the AD mammalian model is unknown. In the present study, we aimed to evaluate the therapeutic potential of D3T in the Tg2576 AD mouse model and investigate the mechanisms underlying its beneficial effects. We showed that intraperitoneal administration of D3T significantly alleviated cognitive deficits in AD mice and dramatically decreased insoluble Aß level and oxidative stress. Further mechanistic studies revealed that D3T significantly promoted hippocampal neurogenesis, and up-regulated levels of silent information regulator 1 (Sirt1), Nrf2 and heme oxygenase-1 (HO-1). Moreover, the positive effect of D3T on behavioral performance of AD mice was markedly attenuated by inhibition of the Sirt1/Nrf2 pathway by the antagonist EX527. In summary, our studies on a mouse AD model indicate that D3T could serve as a potential therapeutic agent for this devastating disease.

Discovery of the leinamycin family of natural products by mining actinobacterial genomes.

Nature's ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. The knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogs by rational metabolic pathway engineering. While successful, structural alterations are limited, with designer analogs often produced in compromised titers. The discovery-based approach to combinatorial biosynthesis complements the knowledge-based approach by exploring the vast combinatorial biosynthesis repertoire found in Nature. Here we showcase the discovery-based approach to combinatorial biosynthesis by targeting the domain of unknown function and cysteine lyase domain (DUF-SH) didomain, specific for sulfur incorporation from the leinamycin (LNM) biosynthetic machinery, to discover the LNM family of natural products. By mining bacterial genomes from public databases and the actinomycetes strain collection at The Scripps Research Institute, we discovered 49 potential producers that could be grouped into 18 distinct clades based on phylogenetic analysis of the DUF-SH didomains. Further analysis of the representative genomes from each of the clades identified 28 lnm-type gene clusters. Structural diversities encoded by the LNM-type biosynthetic machineries were predicted based on bioinformatics and confirmed by in vitro characterization of selected adenylation proteins and isolation and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity and highlight Nature's rich biosynthetic repertoire. Comparative analysis of the LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature's biosynthetic strategies and apply these findings to combinatorial biosynthesis for natural product discovery and structural diversity.

Reduced thione ligation is preferred over neutral phosphine ligation in diiron biomimics regarding electronic functionality: a spectroscopic and computational investigation.

Sulfur containing ligands with a thiolate-thiyl radical character are much superior than phosphine ligands in the active site modeling of [FeFe]hydrogenase regarding electronic functionality on charge communication and modulation of the electronic structure of the catalytic metal center.

Characterization of LnmO as a pathway-specific Crp/Fnr-type positive regulator for leinamycin biosynthesis in Streptomyces atroolivaceus and its application for titer improvement.

The cyclic adenosine monophosphate (cAMP) receptor protein/fumarate and nitrate reductase regulatory protein (Crp/Fnr) family of transcriptional regulators are pleiotropic transcriptional regulators that control a broad range of cellular functions. Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. We previously cloned and characterized the lnm biosynthetic gene cluster from S. atroolivaceus S-140. We here report inactivation of lnmO in S. atroolivaceus S-140 and overexpression of lnmO in the S. atroolivaceus S-140 wild-type and ∆lnmE mutant SB3033 to investigate its role in LNM biosynthesis. Bioinformatics analysis revealed LnmO as the only regulator within the lnm gene cluster, exhibiting high sequence similarity to known Crp/Fnr family regulators. The inactivation of lnmO in S. atroolivaceus S-140 completely abolished LNM production but caused no apparent morphological changes, supporting that LnmO is indispensable and specific to LNM biosynthesis. Overexpression of lnmO in S. atroolivaceus S-140 and SB3033 resulted in three- and fourfold increase in LNM and LNM E1 production, respectively, supporting that LnmO acts as a positive regulator. While all of the Crp/Fnr family regulators studied to date appeared to be pleiotropic, our results support LnmO as the first Crp/Fnr family regulator that is pathway-specific. LnmO joins the growing list of regulators that could be exploited to improve secondary metabolite production in Streptomyces. Engineered strains overproducing LNM and LNM E1 will facilitate further mechanistic studies and clinical evaluation of LNM and LNM E1 as novel anticancer drugs.

Characterization of the Ketosynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase-Polyketide Synthase Interface for Leinamycin Biosynthesis.

Leinamycin (LNM) is biosynthesized by a hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Characterization of LnmI revealed ketosynthase (KS)-acyl carrier protein (ACP)-KS domains at the NRPS-PKS interface. Inactivation of the KS domain or ACP domain in vivo abolished LNM production, and the ACP domain can be phosphopantetheinylated in vitro. The LnmI KS-ACP-KS architecture represents a new mechanism for functional crosstalk between NRPS and AT-less type I PKS in the biosynthesis of hybrid peptide-polyketide natural products.