Yousong Ding

Genome-Based Characterization of Two Prenylation Steps in the Assembly of the Stephacidin and Notoamide Anticancer Agents in a Marine-Derived Aspergillus sp.

Stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a core bicyclo[2.2.2]diazaoctane ring system. These bioactive fungal secondary metabolites have a range of unusual structural and stereochemical features but their biosynthesis has remained uncharacterized. Herein, we report the first biosynthetic gene cluster for this class of fungal alkaloids based on whole genome sequencing of a marine-derived Aspergillus sp. Two central pathway enzymes catalyzing both normal and reverse prenyltransfer reactions were characterized in detail. Our results establish the early steps for creation of the prenylated indole alkaloid structure and suggest a scheme for the biosynthesis of stephacidin and notoamide metabolites. The work provides the first genetic and biochemical insights for understanding the structural diversity of this important family of fungal alkaloids.

Biomimetic Total Synthesis of Malbrancheamide and Malbrancheamide B

The biomimetic total syntheses of both malbrancheamide and malbrancheamide B are reported. The synthesis of the two monochloro species enabled the structure of malbrancheamide B to be unambiguously assigned. The syntheses each feature an intramolecular Diels-Alder reaction of a 5-hydroxypyrazin-2(1H)-one to construct the bicyclo[2.2.2]diazaoctane core, which has also been proposed as the biosynthetic route to these compounds.

Molecular Analysis of a 4-Dimethylallyltryptophan Synthase from Malbranchea aurantiaca

Prenyltransferases are widely distributed in prokaryotes and eukaryotes and play critical roles in cell signaling, protein trafficking, and elaboration of complex molecules in secondary metabolism. Numerous prenylated natural products have been isolated from diverse microorganisms, including bacteria and fungi. These complex metabolites possess a wide range of biological activities, with some showing promise as medicinal agents. On the other hand, many prenylated secondary metabolites have been described as toxins such as ergot alkaloids that have potent psychotropic activity. We have characterized a new prenyltransferase isolated from genomic DNA of Malbranchea aurentiaca RRC1813. Enzyme specificity was investigated with a series of amino acid substrates revealing its function as a 4-dimethylallyltryptophan synthase. Polypeptide sequence alignment analysis showed that it groups with a new class of prenyltransferase enzymes that lack the typical (N/D)DXXD motif found in these polypeptides. MaPT activity was not dependent on a divalent cation cofactor, although it was reversibly inactivated by 5 mm EDTA. Analysis of kinetic parameters showed reduced enzyme efficiency upon simple modification of l-Trp. Moreover, d-Trp had 0.5% relative activity and functioned as a competitive inhibitor with a Ki of 40.41 μm. Finally, Thr-105, Asp-179, Lys-189, and Lys-261 in MaPT were serially mutated, and the resulting lesions displayed low or complete loss of activity. This study provides a detailed characterization of a prenyltransferase in Malbranchea species, reveals two enzyme inhibitors, and through site-directed mutagenesis identified several key amino acid residues in catalysis, yielding new insights into this important yet understudied class of natural product biosynthetic enzymes.

Irisin Controls Growth, Intracellular Ca2+ Signals, and Mitochondrial Thermogenesis in Cardiomyoblasts.

Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin’s potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin) and in HEK293 cells (hr-irisin) for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca2+ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca2+ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work supports the value of exercise, which promotes irisin release.

Chemoenzymatic synthesis of cryptophycin anticancer agents by an ester bond-forming non-ribosomal peptide synthetase module.

Cryptophycins (Crp) are a group of cyanobacterial depsipeptides with activity against drug-resistant tumors. Although they have been shown to be promising, further efforts are required to return these highly potent compounds to the clinic through a new generation of analogues with improved medicinal properties. Herein, we report a chemosynthetic route relying on the multifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety (unit D) into Crp analogues. CrpD-M2 is a unique non-ribosomal peptide synthetase (NRPS) module comprised of condensation-adenylation-ketoreduction-thiolation (C-A-KR-T) domains. We interrogated A-domain 2-keto and 2-hydroxy acid activation and loading, and KR domain activity in the presence of NADPH and NADH. The resulting 2-hydroxy acid was elongated with three synthetic Crp chain elongation intermediate analogues through ester bond formation catalyzed by CrpD-M2 C domain. Finally, the enzyme-bound seco-Crp products were macrolactonized by the Crp thioesterase. Analysis of these sequential steps was enabled through LC-FTICR-MS of enzyme-bound intermediates and products. This novel chemoenzymatic synthesis of Crp involves four sequential catalytic steps leading to the incorporation of a 2-hydroxy acid moiety in the final chain elongation intermediate. The presented work constitutes the first example where a NRPS-embedded KR domain is employed for assembly of a fully elaborated natural product, and serves as a proof-of-principle for chemoenzymatic synthesis of new Crp analogues.

Recent advances in biocatalyst discovery, development and applications

Enzymes catalyze a wide range of biotransformations and have a great potential as environmentally friendly alternatives to classical chemical catalysts in various industrial applications. Recently, advanced techniques and strategies in enzyme discovery and engineering have led to the significant expansion of the quantity and functional diversity of biocatalysts, which has further allowed broader uses of biocatalysts in new processes, especially those traditionally enabled only by chemical catalysts. Here we highlight some of these recent advances with the focus on new approaches in biocatalyst discovery and development, and discuss new applications of selected biocatalysts including transaminases, cytochrome P450s, and Baeyer-Villiger monooxygenases.

Detection of VM55599 and Preparaherquamide from Aspergillus japonicus and Penicillium fellutanum : Biosynthetic Implications

The secondary metabolites VM55599 (4) and preparaherquamide (5) have been identified by LC-MS(n) analysis as natural metabolites in cultures of Penicillium fellutanum, whereas preparaherquamide has been identified only in cultures of Aspergillus japonicus. In accord with a previous proposal, the identification of both metabolites, which have a diastereomeric relationship, provides indirect support for a unified biosynthetic scheme.

Structural basis for precursor protein-directed ribosomal peptide macrocyclization

Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides whose members target proteases with potent reversible inhibition. The product structure is constructed by three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here, we describe the detailed structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases, MdnC and MdnB, interact with a conserved α-helix of the precursor peptide using a novel precursor peptide recognition mechanism. The results provide insight into the unique protein/protein interactions key to the chemistry, suggest an origin of the natural combinatorial synthesis of microviridin peptides and provide a framework for future engineering efforts to generate designed compounds.

Engineered P450 biocatalysts show improved activity and regio-promiscuity in aromatic nitration

Nitroaromatics are among the most important and commonly used chemicals but their production often suffers from multiple unsolved challenges. We have previously described the development of biocatalytic nitration processes driven by an engineered P450 TxtE fusion construct. Herein we report the creation of improved nitration biocatalysts through constructing and characterizing fusion proteins of TxtE with the reductase domain of CYP102A1 (P450BM3, BM3R). The majority of constructs contained variable linker length while one was rationally designed for optimizing protein-protein interactions. Detailed biochemical characterization identified multiple active chimeras that showed improved nitration activity, increased coupling efficiency and higher total turnover numbers compared with TxtE. Substrate promiscuity of the most active chimera was further assessed with a substrate library. Finally, a biocatalytic nitration process was developed to nitrate 4-Me-dl-Trp. The production of both 4-Me-5-NO2-l-Trp and 4-Me-7-NO2-l-Trp uncovered remarkable regio-promiscuity of nitration biocatalysts.

Promiscuous Pathogenicity Islands and Phylogeny of Pathogenic Streptomyces spp.

Approximately 10 Streptomyces species cause disease on underground plant structures. The most economically important of these is potato scab, and the most studied of these pathogens is Streptomyces scabiei (syn. S. scabies). The main pathogenicity determinant of scab-causing Streptomyces species is a nitrated diketopiperazine, known as thaxtomin A (ThxA). In the pathogenic species Streptomyces turgidiscabies, ThxA biosynthetic genes reside on a mobile pathogenicity island (PAI). However, the mobilization of PAIs in other Streptomyces species remains uncharacterized. Here, we investigated the mobilization of the PAI of S. scabiei 87-22. Based on whole genome sequences, we inferred the evolutionary relationships of pathogenic Streptomyces species and discovered that Streptomyces sp. strain 96-12, a novel pathogenic species isolated from potatoes in Egypt, was phylogenetically grouped with nonpathogenic species rather than with known pathogenic species. We also found that Streptomyces sp. strain 96-12 contains a PAI that is almost identical to the PAI in S. scabiei 87-22, despite significant differences in their genome sequences. This suggested direct or indirect in vivo mobilization of the PAI between S. scabiei and nonpathogenic Streptomyces species. To test whether the S. scabiei 87-22 PAI could, indeed, be mobilized, S. scabiei 87-22 deletion mutants containing antibiotic resistance markers in the PAI were mated with Streptomyces diastatochromogenes, a nonpathogenic species. The PAI of S. scabiei was site-specifically inserted into the aviX1 gene of S. diastatochromogenes and conferred pathogenicity in radish seedling assays. Our results demonstrated that S. scabiei, the earliest described Streptomyces pathogen, could be the source of a PAI responsible for the emergence of novel pathogenic species.