Terpene Synthases: One Fold, Many Products

Terpene synthases/cyclases yield core structures of some of the most valuable bioactive molecules, such as artemisinin and paclitaxel. A detailed understanding of structure–function relationship of terpene synthases/cyclases delineates the evolutionary relationship between different enzymes and molecular basis of their strategies to direct and manipulate carbocation intermdiates throughout the catalysis trajectory. Using Aspergillus terreus aristolochene synthase and Streptomyces citricolor germacradiene-4-ol synthase, both of which are high-fidelity cyclases, as model systems, we have provided structural evidence showing that the pyrophosphate group is responsible for carrying out general base–general acid chemistry throughout the carbocation cascade. We also show that despite the highly reactive nature of carbocation intermediates, terpene cyclases accurately guide active site water molecules that are tethered by surrounding residues to serve as a part of the enzymatic template or are catalytically activated at the right time to hydroxylate the final carbocation intermediate. Phomopsis amygdali fusicoccadiene synthase (PaFS) is the first identified bifunctional diterpene synthase that carries out tandem elongation and cyclization reactions. We have shown that cyclization activity of the bona fide substrate geranylgeranyl diphosphate (GGPP) by the full-length enzyme is subject to allosteric regulation, and hexamerization of PaFS driven by the elongation domain creates proximity effects that accelerate product flux by 2 fold. We also present structural studies that shed light on its catalytic mechanism and reveal its evolutionary relationships between bifunctional diterpene synthases and similar sesterterpene synthases

SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis.

Pericyclic reactions-which proceed in a concerted fashion through a cyclic transition state-are among the most powerful synthetic transformations used to make multiple regioselective and stereoselective carbon-carbon bonds. They have been widely applied to the synthesis of biologically active complex natural products containing contiguous stereogenic carbon centres. Despite the prominence of pericyclic reactions in total synthesis, only three naturally existing enzymatic examples (the intramolecular Diels-Alder reaction, and the Cope and the Claisen rearrangements) have been characterized. Here we report a versatile S-adenosyl-l-methionine (SAM)-dependent enzyme, LepI, that can catalyse stereoselective dehydration followed by three pericyclic transformations: intramolecular Diels-Alder and hetero-Diels-Alder reactions via a single ambimodal transition state, and a retro-Claisen rearrangement. Together, these transformations lead to the formation of the dihydropyran core of the fungal natural product, leporin. Combined in vitro enzymatic characterization and computational studies provide insight into how LepI regulates these bifurcating biosynthetic reaction pathways by using SAM as the cofactor. These pathways converge to the desired biosynthetic end product via the (SAM-dependent) retro-Claisen rearrangement catalysed by LepI. We expect that more pericyclic biosynthetic enzymatic transformations remain to be discovered in naturally occurring enzyme 'toolboxes'. The new role of the versatile cofactor SAM is likely to be found in other examples of enzyme catalysis

Cassava genome from a wild ancestor to cultivated varieties

Cassava is a major tropical food crop in the Euphorbiaceae family that has high carbohydrate production potential and adaptability to diverse environments. Here we present the draft genome sequences of a wild ancestor and a domesticated variety of cassava and comparative analyses with a partial inbred line. We identify 1,584 and 1,678 gene models specific to the wild and domesticated varieties, respectively, and discover high heterozygosity and millions of single-nucleotide variations. Our analyses reveal that genes involved in photosynthesis, starch accumulation and abiotic stresses have been positively selected, whereas those involved in cell wall biosynthesis and secondary metabolism, including cyanogenic glucoside formation, have been negatively selected in the cultivated varieties, reflecting the result of natural selection and domestication. Differences in microRNA genes and retrotransposon regulation could partly explain an increased carbon flux towards starch accumulation and reduced cyanogenic glucoside accumulation in domesticated cassava. These results may contribute to genetic improvement of cassava through better understanding of its biology

Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme that Catalyzes C-C Bond Formation

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze various transformations of amino acids at alpha, beta, and gamma positions. These versatile enzymes are prominently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products, and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S, 6S)-6-methyl pipecolate 1, from the biosynthetic pathway of indole alkaloid citrinadin. The key enzyme CndF is PLP-dependent and catalyzes synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed this enzyme performs gamma-elimination of O-acetyl L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new Cgamma-Cdelta bond in 3 and complete the gamma-substitution reaction. CndF displays substrate promiscuity towards different beta-keto carboxylate and esters. Using a recombinant Aspergillus strain expressing CndF and CndE, feeding various alkyl-beta-keto esters led to the biosynthesis of 6-substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes

Biosynthesis of Mycotoxin Fusaric Acid and Application of a PLP-Dependent Enzyme for Chemoenzymatic Synthesis of Substituted l-Pipecolic Acids.

Fusaric acid (FA) is a well-known mycotoxin that plays an important role in plant pathology. The biosynthetic gene cluster for FA has been identified, but the biosynthetic pathway remains unclarified. Here, we elucidated the biosynthesis of FA, which features a two-enzyme catalytic cascade, a pyridoxal 5-phosphate (PLP)-dependent enzyme (Fub7), and a flavin mononucleotide (FMN)-dependent oxidase (Fub9) in synthesizing the picolinic acid scaffold. FA biosynthesis also involves an off-line collaboration between a highly reducing polyketide synthase (HRPKS, Fub1) and a nonribosomal peptide synthetase (NRPS)-like carboxylic acid reductase (Fub8) in making an aliphatic α,β-unsaturated aldehyde. By harnessing the stereoselective C-C bond-forming activity of Fub7, we established a chemoenzymatic route for stereoconvergent synthesis of a series of 5-alkyl-, 5,5-dialkyl-, and 5,5,6-trialkyl-l-pipecolic acids of high diastereomeric ratio

Biosynthesis of Mycotoxin Fusaric Acid and Application of a PLP-Dependent Enzyme for Chemoenzymatic Synthesis of Substituted L-Pipecolic Acids

Fusaric acid (FA) is a well-known mycotoxin that plays an important role in plant pathology. The biosynthetic gene cluster for FA has been identified but the biosynthetic pathway remains unclarified. Here, we elucidated the biosynthesis of FA, which features a two-enzyme catalytic cascade, a pyridoxal 5’-phosphate (PLP)-dependent enzyme (Fub7) and a flavin mononucleotide (FMN)-dependent oxidase (Fub9) in synthesizing the picolinic acid scaffold. FA biosynthesis also involves an off-line collaboration between a highly reducing polyketide synthase (HRPKS, Fub1) and a nonribosomal peptide synthetase (NRPS)-like carboxylic acid reductase (Fub8) in making an aliphatic alpha,beta-unsaturated aldehyde. By harnessing the stereoselective C-C bond forming activity of Fub7, we established a chemoenzymatic route for stereoconvergent synthesis of a series of 5-alkyl, 5,5-dialkyl, and 5,5,6-trialkyl-L-pipecolic acids of high diastereomeric ratio. </div

Research progress of carbon based nanoenzyme and composites in antibacterial field

Due to the abuse of antibiotics, more and more bacteria are resistant to antibiotics. Non antibiotic nano antibacterial materials emerge as the times require. Carbon based nano enzyme is an efficient and environmentally friendly antibacterial material with certain antibacterial effect. It has simple structure and good compatibility. It can be combined with a variety of antibacterial substances to form composite antibacterial materials, expand the scope of antibacterial and improve the antibacterial ability. This paper summarizes the research progress of three kinds of carbon based nanoenzymes including carbon nanotubes, graphene, carbon quantum dots and their composites in the field of antibacterial

Coevolutionary Dynamics of Actions and Opinions in Social Networks

Modeling opinion formation and decision-making processes, important in their own rights, have been treated as separate problems in the study of dynamical models for social networks. Empirical studies suggest a deep intertwining between these two processes, and in this paper, we bridge the gap in the existing research by proposing a novel coevolutionary model. In the model, each individual can select an action from a binary set, and also holds an opinion on which action they prefer. Actions and opinions coevolve on a two-layer network structure. Under some reasonable assumptions on the network structure and asynchronous updating mechanics, we use rigorous analysis to establish that for all initial conditions, the actions converge in a finite number of time steps while opinions converge asymptotically. Next, we provide sufficient conditions for the emergence and the stability of polarized equilibria, whereby the population splits into two communities, each selecting and supporting one of the actions. Finally, numerical simulations are used to examine "pluralistic ignorance", whereby a social group incorrectly assumes the opinions of others due to the actions observed.Comment: Manuscript submitted to IEEE Transactions on Automatic Control 2021 August 9t