Simulation-Guided Engineering Enables a Functional Switch in Selinadiene Synthase toward Hydroxylation.

Engineering sesquiterpene synthases to form predefined alternative products is a major challenge due to their diversity in cyclization mechanisms and our limited understanding of how amino acid changes affect the steering of these mechanisms. Here, we use a combination of atomistic simulation and site-directed mutagenesis to engineer a selina-4(15),7(11)-diene synthase (SdS) such that its final reactive carbocation is quenched by trapped active site water, resulting in the formation of a complex hydroxylated sesquiterpene (selin-7(11)-en-4-ol). Initially, the SdS G305E variant produced 20% selin-7(11)-en-4-ol.

Single Point Mutation Abolishes Water Capture in Germacradien-4-ol Synthase.

The high-fidelity sesquiterpene cyclase (-)-germacradien-4-ol synthase (GdolS) converts farnesyl diphosphate into the macrocyclic alcohol (-)-germacradien-4-ol. Site-directed mutagenesis was used to decipher the role of key residues in the water control mechanism. Replacement of Ala176, located in the G1/2 helix, with non-polar aliphatic residues of increasing size (valine, leucine, isoleucine and methionine) resulted in the accumulation of the non-hydroxylated products germacrene A and germacrene D.

Production of non-natural terpenoids through chemoenzymatic synthesis using substrate analogs.

Chemoenzymatic synthesis of non-natural terpenes using the promiscuous activity of terpene synthases allows for the expansion of the chemical space of terpenoids with potentially new bioactivities. In this report, we describe protocols for the preparation of a novel aphid attractant, (S)-14,15-dimethylgermacrene D, by exploiting the promiscuity of (S)-germacrene D synthase from Solidago canadensis and using an engineered biocatalytic route to convert prenols to terpenoids. The method uses a combination of five enzymes to carry out the preparation of terpenoid semiochemicals in two steps: (1) diphosphorylation of five or six carbon precursors (prenol, isoprenol and methyl-isoprenol) catalyzed by Plasmodium falciparum choline kinase and Methanocaldococcus jannaschii isopentenyl phosphate kinase to form DMADP, IDP and methyl-IDP, and (2) chain elongation and cyclization catalyzed by Geobacillus stearothermophilus (2E,6E)-farnesyl diphosphate synthase and S.

Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases.

Terpene synthases (TS) catalyze complex reactions to produce a diverse array of terpene skeletons from linear isoprenyl diphosphates. Patchoulol synthase (PTS) from converts farnesyl diphosphate into patchoulol. Using simulation-guided engineering, we obtained PTS variants that eliminate water capture.

Redesigning the Molecular Choreography to Prevent Hydroxylation in Germacradien-11-ol Synthase Catalysis.

Natural sesquiterpene synthases have evolved to make complex terpenoids by quenching reactive carbocations either by proton transfer or by hydroxylation (water capture), depending on their active site. Germacradien-11-ol synthase (Gd11olS) from catalyzes the cyclization of farnesyl diphosphate (FDP) into the hydroxylated sesquiterpene germacradien-11-ol. Here, we combine experiment and simulation to guide the redesign of its active site pocket to avoid hydroxylation of the product.

Genome-wide discovery of OsHOX24-binding sites and regulation of desiccation stress response in rice.

OsHOX24 mediates regulation of desiccation stress response via complex regulatory network as indicated by its binding to several target genes including transcription factors in rice. HD-ZIP I subfamily of homeobox transcription factors (TFs) are involved in abiotic stress responses and plant development. Previously, we demonstrated the role of OsHOX24, a member of HD-ZIP I subfamily, in abiotic stress responses.

Therapeutic potential of cyanobacterial pigment protein phycoerythrin: in silico and in vitro study of BACE1 interaction and in vivo Aβ reduction.

Cyanobacteria are an immense source of innovative classes of pharmacologically active compounds exhibiting various biological activities ranging from antioxidants, antibiotics, anticancer, anti-inflammatory to anti-Alzheimer's disease. In the present study, we primarily targeted the inhibition of Beta-site amyloid precursor protein cleaving enzyme-1 (BACE1) by a naturally occurring cyanobacterial protein phycoerythrin (C-PE). BACE1 cleaves amyloid-β precursor protein (APP) and leads to accumulation of neurotoxic amyloid beta (Aβ) plaques in the brain, as an attribute of Alzheimer's disease (AD).

Comprehensive metabolic and transcriptomic profiling of various tissues provide insights for saponin biosynthesis in the medicinally important Asparagus racemosus.

Asparagus racemosus (Shatavari), belongs to the family Asparagaceae and is known as a "curer of hundred diseases" since ancient time. This plant has been exploited as a food supplement to enhance immune system and regarded as a highly valued medicinal plant in Ayurvedic medicine system for the treatment of various ailments such as gastric ulcers, dyspepsia, cardiovascular diseases, neurodegenerative diseases, cancer, as a galactogogue and against several other diseases. In depth metabolic fingerprinting of various parts of the plant led to the identification of 13 monoterpenoids exclusively present in roots.

De novo transcriptome assembly and comprehensive expression profiling in Crocus sativus to gain insights into apocarotenoid biosynthesis.

Saffron (Crocus sativus L.) is commonly known as world's most expensive spice with rich source of apocarotenoids and possesses magnificent medicinal properties. To understand the molecular basis of apocarotenoid biosynthesis/accumulation, we performed transcriptome sequencing from five different tissues/organs of C.

Functional Characterization of Novel Sesquiterpene Synthases from Indian Sandalwood, Santalum album.

Indian Sandalwood, Santalum album L. is highly valued for its fragrant heartwood oil and is dominated by a blend of sesquiterpenes. Sesquiterpenes are formed through cyclization of farnesyl diphosphate (FPP), catalyzed by metal dependent terpene cyclases.

Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis.

Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial.

Biocatalyst mediated regio- and stereo-selective hydroxylation and epoxidation of (Z)-α-santalol.

Biocatalyst mediated regio- and stereo-selective hydroxylation and epoxidation on (Z)-α-santalol were achieved for the first time, using a fungal strain Mucor piriformis. Four novel metabolites were characterized as 10,11-cis-β-epoxy-α-santalol, 5α-hydroxy-(Z)-α-santalol, 10,11-dihydroxy-α-santalol and 5α-hydroxy-10,11-cis-β-epoxy-α-santalol. Using Amano PS lipase from Burkholderia cepacia, α- and β-isomers of 10,11-cis-epoxy-α-santalol were resolved efficiently.

Self-assembly to function: design, synthesis, and broad spectrum antimicrobial properties of short hybrid E-vinylogous lipopeptides.

Nonribosomal E-vinylogous γ-amino acids are widely present in many peptide natural products and have been exploited as inhibitors for serine and cysteine proteases. Here, we are reporting the broad spectrum antimicrobial properties and self-assembled nanostructures of various hybrid lipopeptides composed of 1:1 alternating α- and E-vinylogous residues. Analysis of the results revealed that self-assembled nanostructures also play a significant role in the antimicrobial and hemolytic activities.

Preparative separation of α- and β-santalenes and (Z)-α- and (Z)-β-santalols using silver nitrate-impregnated silica gel medium pressure liquid chromatography and analysis of sandalwood oil.

The major sesquiterpene constituents of East-Indian sandalwood oil (Z)-α- and (Z)-β-santalols have shown to be responsible for most of the biological activities and organoleptic properties of sandalwood oil. The work reported here describes the strategic use of medium pressure liquid chromatography (MPLC) for the separation of both α- and β-santalenes and (Z)-α- and (Z)-β-santalols. Silver nitrate impregnated silica gel was used as the stationary phase in MPLC for quantitative separation of α- and β-santalenes and (Z)-α- and (Z)-β-santalols with mobile phases hexane and dichloromethane, respectively.