Combating Staphylococcus aureus biofilm formation: the inhibitory potential of tormentic acid and 23-hydroxycorosolic acid.

Plant extracts have been used to treat microbiological diseases for centuries. This study examined plant triterpenoids tormentic acid (TA) and 23-hydroxycorosolic acid (HCA) for their antibiofilm effects on Staphylococcus aureus strains (MTCC-96 and MTCC-7405). Biofilms are bacterial colonies bound by a matrix of polysaccharides, proteins, and DNA, primarily impacting healthcare. As a result, ongoing research is being conducted worldwide to control and prevent biofilm formation. Our research showed that TA and HCA inhibit S. aureus planktonic growth by depolarizing the bacterial membrane. In addition, zone of inhibition studies confirmed their effectiveness, and crystal violet staining and biofilm protein quantification confirmed their ability to prevent biofilm formation. TA and HCA exhibited substantial reductions in biofilm formation for S. aureus (MTCC-96) by 54.85% and 48.6% and for S. aureus (MTCC-7405) by 47.07% and 56.01%, respectively. Exopolysaccharide levels in S. aureus biofilm reduced significantly by TA (25 µg/mL) and HCA (20 µg/mL). Microscopy, bacterial motility, and protease quantification studies revealed their ability to reduce motility and pathogenicity. Furthermore, TA and HCA treatment reduced the mRNA expression of S. aureus virulence genes. In silico analysis depicted a high binding affinity of triterpenoids for biofilm and quorum-sensing associated proteins in S. aureus, with TA having the strongest affinity for TarO (- 7.8 kcal/mol) and HCA for AgrA (- 7.6 kcal/mol). TA and HCA treatment reduced bacterial load in S. aureus-infected peritoneal macrophages and RAW264.7 cells. Our research indicates that TA and HCA can effectively combat S. aureus by inhibiting its growth and suppressing biofilm formation.

The anti-biofilm potential of triterpenoids isolated from Sarcochlamys pulcherrima (Roxb.) Gaud.

Formation of biofilm is the major cause of Pseudomonas aeruginosa associated pathological manifestations in the urinary tract, respiratory system, gastrointestinal tract, skin, soft tissues etc. Triterpenoid group of compounds have shown their potential in reducing planktonic and biofilm form of bacteria. Sarcochlamys pulcherrima (Roxb.) Gaud. is an ethnomedicinal plant traditionally used for its anti-microbial and anti-inflammatory property. In the present study two triterpenoids, have been isolated from this plant, characterised and evaluated for their antibacterial and antibiofilm potential against P. aeruginosa. Compounds were characterised as 2α, 3ß, 19α-trihydroxy-urs-12-ene-28-oic acid (Tormentic acid) and 2α, 3ß, 23-trihydroxyurs-12-ene-28-oic acid (23-hydroxycorosolic acid) through spectroscopic studies viz. infrared (IR), nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Depolarization of bacterial membrane and zone of inhibition studies revealed that both the compounds inhibited the growth of planktonic bacteria. Compounds were also found to inhibit the formation of P. aeruginosa biofilm. Inhibition of biofilm found to be mediated through suppressed secretion of pyoverdin, protease and swarming motility of P. aeruginosa. Gene expression study, in silico binding analysis, in vivo bacterial load and tissue histology observations also supported the antibiofilm activity of both the compounds. In vitro and in vivo study showed that both compounds were non-toxic. The study has explored the antibacterial and antibiofilm effect of two triterpenes isolated for the first time from S. pulcherrima.

Identification of a unique loss-of-function mutation in IGF1R and a crosstalk between IGF1R and Wnt/ß-catenin signaling pathways.

IGF1R is a ubiquitous receptor tyrosine kinase that plays critical roles in cell proliferation, growth and survival. Clinical studies have demonstrated upregulation of IGF1R mediated signaling in a number of malignancies including colon, breast, and lung cancers. Overexpression of the IGF1R in these malignancies is associated with a poor prognosis and overall survival. IGF1R specific kinase inhibitors have failed in multiple clinical trials partly because of the complex nature of IGF1R signaling. Thus identifying new binding partners and allosteric sites on IGF1R are emerging areas of research. More recently, IGF1R has been shown to translocate into the nucleus and perform many functions. In this study, we generated a library of IGF1R deletion and point mutants to examine IGF1R subcellular localization and activation of downstream signaling pathways. We show that the nuclear localization of IGF1R is primarily defined by its cytoplasmic domain. We identified a cross-talk between IGF1R and Wnt/ß-catenin signaling pathways and showed, for the first time, that IGF1R is associated with upregulation of TCF-mediated ß-catenin transcriptional activity. Using loss-of-function mutants, deletion analysis and IGF1R specific inhibitor(s), we show that cytoplasmic and nuclear activities are two independent functions of IGF1R. Furthermore, we identified a unique loss-of-function mutation in IGF1R. This unique loss-of-function mutant retains only nuclear functions and sits in a pocket, outside ATP and substrate binding region, that is suited for designing allosteric inhibitors of IGF1R.

Attenuation of neuroblastoma cell growth by nisin is mediated by modulation of phase behavior and enhanced cell membrane fluidity.

Antimicrobial peptides have been attracting significant attention as potential anti-cancer therapeutic agents in recent times. Yet most antimicrobial peptides seem to possess cytotoxic effects on non-cancerous cells. Nisin, an antimicrobial peptide and FDA approved food preservative, has recently been found to induce selective apoptotic cell death and reduced cell proliferation in different cancer cell lines. However, the mechanism of nisin interaction with cancer cell membranes remains unexplored. Using potentiometric dye-based fluorescence and monolayer surface pressure-area isotherms we find that nisin interaction enhances the fluidity and reduces the dipole potential of a neuroblastoma cell membrane model. The quantified compressibility modulus suggests that the changes in fluidity are predominantly driven by the nisin interaction with the non-raft like regions. However, the measured positive Gibbs free energy of mixing and enthalpy hints that nisin, owing to its unfavorable mixing with cholesterol, might significantly disrupt the raft-like domains.

Evolution of structural fitness and multifunctional aspects of mycobacterial RND family transporters.

Drug resistance is a major concern due to the evolution and emergence of pathogenic bacterial strains with novel strategies to resist the antibiotics in use. Mycobacterium tuberculosis (Mtb) is one of such pathogens with reported strains, which are not treatable with any of the available anti-TB drugs. This scenario has led to the need to look for some novel drug targets in Mtb, which may be exploited to design effective treatment strategies against the infection. The goal of this review is to discuss one such class of emerging drug targets in Mtb. MmpL (mycobacterial membrane protein large) proteins from Mtb are reported to be involved in multi-substrate transport including drug efflux and considered as one of the contributing factors for the emergence of multidrug-resistant strains. MmpL proteins belong to resistance nodulation division permeases superfamily of membrane transporters, which are viably and pathogenetically important and their inhibition could be lethal for the bacteria.

Modulation of S. aureus and P. aeruginosa biofilm: an in vitro study with new coumarin derivatives.

Coumarin is an important heterocyclic molecular framework of bioactive molecules against broad spectrum pathological manifestations. In the present study 18 new coumarin derivatives (CDs) were synthesized and characterized for antibiofilm activity against two model bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. It was observed that all the CDs executed significant effect in moderating activities against both planktonic and biofilm forms of these selected bacteria. Hence, to interpret the underlying probable reason of such antibiofilm effect, in-silico binding study of CDs with biofilm and motility associated proteins of these organisms were performed. All CDs have shown their propensity for occupying the native substrate binding pocket of each protein with moderate to strong binding affinities. One of the CDs such as CAMN1 showed highest binding affinity with these proteins. Interestingly, the findings of in-silico studies coincides the experimental results of antibiofilm and motility affect of CDs against both S. aureus and P. aeruginosa. Moreover, in-silico studies suggested that the antibiofilm activity of test CDs may be due to the interference of biofilm and motility associated proteins of the selected model organisms (PilT from P. aeruginosa and TarK, TarO from S. aureus). The detailed synthesis, characterization, methodology and results of biological screening along with computational studies have been reported. This study could be of greater interest in the context of the development of new anti-bacterial agent in the future.

Genome scale identification, structural analysis, and classification of periplasmic binding proteins from Mycobacterium tuberculosis.

Periplasmic-binding proteins occupy the periplasmic space of bacteria and are involved in binding and transport of various ions, siderophores, and other diverse types of solutes. These proteins may be associated with membrane transport systems or may help in activation of signal transducers. There is limited information available on Mycobacterium tuberculosis (Mtb) periplasm-inhabiting proteins. In the present study, we have performed genome-wide identification and functional annotation of periplasmic-binding proteins of Mtb on the basis of signature characteristics and their functional motifs. 37 putative periplasmic-binding proteins were identified in Mtb proteome and categorized into different classes mainly known for their association with membrane transport and signaling pathways. Conclusively, this study adds 11 completely novel proteins to the periplasmic binding proteome of Mtb, which were not annotated as PBPs earlier. This study provides an overview of the periplasmic binding proteome of Mtb, which may be involved in various important patho-physiological functions of the bacteria. These proteins may serve as novel drug targets, which may lead to better treatment strategies against this deadly pathogen.

Exploration of Phytoconstituents from Mussaenda roxburghii and Studies of Their Antibiofilm Effect.

In the context of ethno botanical importance with no phytochemical investigations, Mussaenda roxburghii have been investigated to explore it's phytoconstituents and studies of their antibiofilm activity. Four compounds have been isolated from the aerial parts of this plant and were characterized as 2α,3ß,19α,23-tetrahydroxyurs-12-en-28-oic acid (1), ß-sitosterol glucoside (4), lupeol palmitate (5), and myoinositol (6). All these compounds were tested for antibacterial and antibiofilm activity against Pseudomonas aeruginosa. Compound 1 exhibited three times more antibiofilm activity with minimum inhibitory concentration (MIC) at 0.74 mm compared to that of streptomycin. Molecular docking studies exhibited a very high binding affinity of 1 with P. aeruginosa quorum sensing proteins and motility associated proteins viz. LasR and PilB, PilY1, PilT, respectively. Compound 1 was also found to be non-cytotoxic against sheep RBC and murine peritoneal macrophages at selected sub-MIC doses.

The drug binding sites and transport mechanism of the RND pumps from Mycobacterium tuberculosis: Insights from molecular dynamics simulations.

RND permease superfamily drug efflux pumps are involved in multidrug transport and are attractive to study them for therapeutic purpose. In previous work we have classified 14 members of MmpL proteins belong to RND superfamily from Mycobacterium tuberculosis (Mtb) within its families [Sandhu P. and Akhter Y., 2015. Int. J. Med. Microbiol., 305:413-423]. In this study, structures of these proteins are homology modelled. The drug binding sites and channels are identified using local micro-stereochemistry and charge densities. Potential transport mechanism based on differential structural behaviour in the absence and on the binding of drug molecules is explained using the molecular dynamics simulation results. Our studies show two potential drug binding sites positioned at opposite ends of the transport tunnel leading from cytoplasmic to the periplasmic space across MmpL5 trimer. The drug binding have effects on the structural conformation of the protein leading to molecular-scale peristaltic movements. The free binding energy calculations reveal that the subsequent binding events are interdependent and may have implications on transport mechanism. Two drug binding sites and a continuous channel in the RND pump have been reported. The proposed ligand binding mechanism shows peristaltic movements in the channel leading to the drug efflux. This study would be helpful in understanding the molecular basis of drugs resistance in Mtb.

The internal gene duplication and interrupted coding sequences in the MmpL genes of Mycobacterium tuberculosis: Towards understanding the multidrug transport in an evolutionary perspective.

The multidrug resistance has emerged as a major problem in the treatment of many of the infectious diseases. Tuberculosis (TB) is one of such disease caused by Mycobacterium tuberculosis. There is short term chemotherapy to treat the infection, but the main hurdle is the development of the resistance to antibiotics. This resistance is primarily due to the impermeable mycolic acid rich cell wall of the bacteria and other factors such as efflux of antibiotics from the bacterial cell. The MmpL (Mycobacterial Membrane Protein Large) proteins of mycobacteria are involved in the lipid transport and antibiotic efflux as indicated by the preliminary reports. We present here, comprehensive comparative sequence and structural analysis, which revealed topological signatures shared by the MmpL proteins and RND (Resistance Nodulation Division) multidrug efflux transporters. This provides evidence in support of the notion that they belong to the extended RND permeases superfamily. In silico modelled tertiary structures are in homology with an integral membrane component present in all of the RND efflux pumps. We document internal gene duplication and gene splitting events happened in the MmpL genes, which further elucidate the molecular functions of these putative transporters in an evolutionary perspective.

Vitexin alters Staphylococcus aureus surface hydrophobicity to obstruct biofilm formation.

Cell Surface hydrophobicity is one of the determinant biophysical parameters of bacterial aggregation for being networked to form a biofilm. Phytoconstituent, like vitexin, has long been in use for their antibacterial effect. The present work demonstrates the role of vitexin in modulating Staphylococcus aureus surface hydrophobicity while aggregating to form biofilm and pathogenesis in a host. In planktonic form, vitexin shows minimum inhibitory concentration at 252 µg/ml against S. aureus. Sub-MIC doses of vitexin and antibiotics (26 µg/ml of vitexin, 55 µg/ml of azithromycin, and 2.5 µg/ml of gentamicin) were selected to treat S. aureus. Dead cell counts after treatment were studied through flow cytometry. As dead cell counts were minimal (<5 %), these doses were considered for all subsequent experiments. While studying aggregating cells, it was observed that vitexin reduces S. aureus surface hydrophobicity and membrane permeability at the sub-MIC dose of 26 µg/ml. The in silico binding analysis showed a higher binding affinity of vitexin with surface proteins (IcaA, DltA, and SasG) of S. aureus. Down-regulation of dltA and icaAB expression, along with the reduction in membrane potential with a sub-MIC dose of vitexin, explains reduced S. aureus surface hydrophobicity. Vitexin was found to interfere with S. aureus biofilm-associated protein biomass, EPS production, and swarming movement. Subsequently, the suppression of proteases production and down-regulation of icaAB and agrAC gene expression with a sub-MIC dose of vitexin explained the inhibition of S. aureus virulence in vitro. Besides, vitexin was also found to potentiate the antibiofilm activity of sub-MIC doses of gentamicin and azithromycin. Treatment with vitexin exhibits a protective response in S. aureus infected macrophages through modulation of expression of cytokines like IL-10 and IL-12p40 at protein and mRNA levels. Furthermore, CFU count and histological examination of infected mouse tissue (liver and spleen) justify the in vivo protective effect of vitexin from S. aureus biofilm-associated infection. From this study, it can be inferred that vitexin can reduce S. aureus surface hydrophobicity, leading to interference with aggregation at the time of biofilm formation and subsequent pathogenesis in a host.

Role of allosteric switches and adaptor domains in long-distance cross-talk and transient tunnel formation.

Transient tunnels that assemble and disassemble to facilitate passage of unstable intermediates in enzymes containing multiple reaction centers are controlled by allosteric cues. Using the 140-kDa purine biosynthetic enzyme PurL as a model system and a combination of biochemical and x-ray crystallographic studies, we show that long-distance communication between ~25-Å distal active sites is initiated by an allosteric switch, residing in a conserved catalytic loop, adjacent to the synthetase active site. Further, combinatory experiments seeded from molecular dynamics simulations help to delineate transient states that bring out the central role of nonfunctional adaptor domains. We show that carefully orchestrated conformational changes, facilitated by interplay of dynamic interactions at the allosteric switch and adaptor-domain interface, control reactivity and concomitant formation of the ammonia tunnel. This study asserts that substrate channeling is modulated by allosteric hotspots that alter protein energy landscape, thereby allowing the protein to adopt transient conformations paramount to function.

Analyzing structural differences between insulin receptor (IR) and IGF1R for designing small molecule allosteric inhibitors of IGF1R as novel anti-cancer agents.

IR and insulin-like growth factor-1 receptor (IGF-1R) share high degree of sequence and structural similarity that hinders the development of anticancer drugs targeting IGF1R, which is dysregulated in many cancers. Although IR and IGF1R mediate their activities through similar signalling pathways, yet they show different physiological effects. The exact molecular mechanism(s) how IR and IGF1R exert their distinct functions remain largely unknown. Here, we performed in silico analysis and generated GFP-fusion proteins of wild type IR and its K1079R mutant to analyze their subcellular localization, cytoplasmic and nuclear activities in comparison to IGF1R and its K1055R mutant. We showed that, like K1055R mutation in IGF1R, K1079R mutation does not impede the subcellular localization and nuclear activities of IR. Although K1079R mutation significantly decreases the kinase activity of IR but not as much as K1055R mutation, which was seen to drastically reduce the kinase activity of IGF1R. Moreover, K1079 residue in IR is seen to be sitting in a pocket which is different than the allosteric inhibitor binding pocket present in its homologue (IGF1R). This is for the first time such a study has been conducted to identify structural differences between these receptors that could be exploited for designing small molecule allosteric inhibitor(s) of IGF1R as novel anti-cancer drugs.

Siderophore transport by MmpL5-MmpS5 protein complex in Mycobacterium tuberculosis.

Iron is an essential metal ion required for the various physiological activities of bacteria. The pathogenic bacteria remain dependent on the host cell for their iron requirements and evolved with specialized scavenging machinery in the form of iron chelating siderophores. Mycobacterium tuberculosis has two types of siderophore molecules, mycobactin and carboxymycobactin. These are synthesized inside bacterial cells and need to be transported outside by specialized membrane associated proteins. MmpL5-MmpS5 (mycobacterial membrane protein large5-mycobacterial membrane protein small5) complex has been linked to the export of non-ferrated siderophores to extracellular environment but the precise molecular mechanism involved was largely unknown. We have investigated the association of MmpL5 with mycobactin synthesis and transport associated proteins using system wide protein-protein interaction network. Insights of mycobactin transport mechanism by MmpL5-MmpS5 complex was explored using docking and molecular dynamics simulations. The MmpL5 has association with many proteins with reported roles in iron acquisition or mycobactin biosynthesis. The molecular dynamics simulation analysis after mycobactin docking into MmpL5 binding pockets showed that at cytoplasmic binding site, mycobactin could move towards the central channel of efflux pump and at periplasmic binding site towards the periplasm. MmpL5 was observed to carry out uptake of mycobactin from the cytoplasm and its release into the periplasmic space and MmpS5 was found to facilitate the periplasmic release of mycobactin and enhancement in the transport function of MmpL5. The mycobactin export is an attractive target for drug discovery and it may be carried out by inhibiting the MmpL5 protein's transport function.

Molecular Dynamics Simulations, Challenges and Opportunities: A Biologist's Prospective.

Molecular dynamics (MD) is a computational technique which is used to study biomolecules in virtual environment. Each of the constituent atoms represents a particle and hence the biomolecule embodies a multi-particle mechanical system analyzed within a simulation box during MD analysis. The potential energies of the atoms are explained by a mathematical expression consisting of different forces and space parameters. There are various software and force fields that have been developed for MD studies of the biomolecules. MD analysis has unravelled the various biological mechanisms (protein folding/unfolding, protein-small molecule interactions, protein-protein interactions, DNA/RNA-protein interactions, proteins embedded in membrane, lipid-lipid interactions, drug transport etc.) operating at the atomic and molecular levels. However, there are still some parameters including torsions in amino acids, carbohydrates (whose structure is extended and not well defined like that of proteins) and single stranded nucleic acids for which the force fields need further improvement, although there are several workers putting in constant efforts in these directions. The existing force fields are not efficient for studying the crowded environment inside the cells, since these interactions involve multiple factors in real time. Therefore, the improved force fields may provide the opportunities for their wider applications on the complex biosystems in diverse cellular conditions. In conclusion, the intervention of MD in the basic sciences involving interdisciplinary approaches will be helpful for understanding many fundamental biological and physiological processes at the molecular levels that may be further applied in various fields including biotechnology, fisheries, sustainable agriculture and biomedical research.

Structural basis of transport function in major facilitator superfamily protein from Trichoderma harzianum.

Trichothecenes are the sesquiterpenes secreted by Trichoderma spp. residing in the rhizosphere. These compounds have been reported to act as plant growth promoters and bio-control agents. The structural knowledge for the transporter proteins of their efflux remained limited. In this study, three-dimensional structure of Thmfs1 protein, a trichothecene transporter from Trichoderma harzianum, was homology modelled and further Molecular Dynamics (MD) simulations were used to decipher its mechanism. Fourteen transmembrane helices of Thmfs1 protein are observed contributing to an inward-open conformation. The transport channel and ligand binding sites in Thmfs1 are identified based on heuristic, iterative algorithm and structural alignment with homologous proteins. MD simulations were performed to reveal the differential structural behaviour occurring in the ligand free and ligand bound forms. We found that two discrete trichothecene binding sites are located on either side of the central transport tunnel running from the cytoplasmic side to the extracellular side across the Thmfs1 protein. Detailed analysis of the MD trajectories showed an alternative access mechanism between N and C-terminal domains contributing to its function. These results also demonstrate that the transport of trichodermin occurs via hopping mechanism in which the substrate molecule jumps from one binding site to another lining the transport tunnel.

Proteome scale identification, classification and structural analysis of iron-binding proteins in bread wheat.

Bread wheat is one of the major staple foods of worldwide population and iron plays a significant role in growth and development of the plant. In this report, we are presenting the genome wide identification of iron-binding proteins in bread wheat. The wheat genome derived putative proteome was screened for identification of iron-binding sequence motifs. Out of 602 putative iron-binding proteins, 130 were able to produce reliable structural models by homology techniques and further analyzed for the presence of iron-binding structural motifs. The computationally identified proteins appear to bind to ferrous and ferric ions and showed diverse coordination geometries. Glu, His, Asp and Cys amino acid residues were found to be mostly involved in iron binding. We have classified these proteins on the basis of their localization in the different cellular compartments. The identified proteins were further classified into their protein folds, families and functional classes ranging from structure maintenance of cellular components, regulation of gene expression, post translational modification, membrane proteins, enzymes, signaling and storage proteins. This comprehensive report regarding structural iron binding proteome provides useful insights into the diversity of iron binding proteins of wheat plants and further utilized to study their roles in plant growth, development and physiology.

Antileishmanial and immunomodulatory activities of lupeol, a triterpene compound isolated from Sterculia villosa.

Visceral leishmaniasis (VL) is one of the most severe forms of leishmaniasis, caused by the protozoan parasite Leishmania donovani. Nowadays there is a growing interest in the therapeutic use of natural products to treat parasitic diseases. Sterculia villosa is an ethnomedicinally important plant. A triterpenoid was isolated from this plant and was screened for its antileishmanial and immunomodulatory activities in vitro and in vivo. Biochemical colour test and spectroscopic data confirmed that the isolated pure compound was lupeol. Lupeol exhibited significant antileishmanial activity, with IC50 values of 65 ± 0.41 µg/mL and 15 ± 0.45 µg/mL against promastigote and amastigote forms, respectively. Lupeol caused maximum cytoplasmic membrane damage of L. donovani promastigote at its IC50 dose. It is well known that during infection the Leishmania parasite exerts its pathogenicity in the host by suppressing nitric oxide (NO) production and inhibiting pro-inflammatory responses. It was observed that lupeol induces NO generation in L. donovani-infected macrophages, followed by upregulation of pro-inflammatory cytokines and downregulation of anti-inflammatory cytokines. Lupeol was also found to reduce the hepatic and splenic parasite burden through upregulation of the pro-inflammatory response in L. donovani-infected BALB/c mice. Strong binding affinity of lupeol was observed for four major potential drug targets, namely pteridine reductase 1, adenine phosphoribosyltransferase, lipophosphoglycan biosynthetic protein and glycoprotein 63 of L. donovani, which also supported its antileishmanial and immunomodulatory activities. Therefore, the present study highlights the antileishmanial and immunomodulatory activities of lupeol in an in vitro and in vivo model of VL.

Proteome scale census of major facilitator superfamily transporters in Trichoderma reesei using protein sequence and structure based classification enhanced ranking.

Trichoderma spp. have been acknowledged as potent bio-control agents against microbial pathogens and also as plant growth promoters. Various secondary metabolites are attributed for these beneficial activities. Major facilitator superfamily (MFS) includes the large proportion of efflux-pumps which are linked with membrane transport of these secondary metabolites. We have carried out a proteome-wide identification of MFS transporters using protein sequence and structure based hierarchical method in Trichoderma reesei. 448 proteins out of 9115 were detected to carry transmembrane helices. MFS specific intragenic gene duplication and its context with transport function have been presented. Finally, using homology based techniques, domains and motifs of MFS families have been identified and utilized to classify them. From query dataset of 448 transmembrane proteins, 148 proteins are identified as potential MFS transporters. Sugar porter, drug: H(+) antiporter-1, monocarboxylate porter and anion: cation symporter emerged as major MFS families with 51, 35, 17 and 11 members respectively. Representative protein tertiary structures of these families are homology modeled for structure-function analysis. This study may help to understand the molecular basis of secretion and transport of agriculturally valuable secondary metabolites produced by these bio-control fungal agents which may be exploited in future for enhancing its biotechnological applications in eco-friendly sustainable development.

Structural and mechanistic analysis of engineered trichodiene synthase enzymes from Trichoderma harzianum: towards higher catalytic activities empowering sustainable agriculture.

Trichoderma spp. are well-known bioagents for the plant growth promotion and pathogen suppression. The beneficial activities of the fungus Trichoderma spp. are attributed to their ability to produce and secrete certain secondary metabolites such as trichodermin that belongs to trichothecene family of molecules. The initial steps of trichodermin biosynthetic pathway in Trichoderma are similar to the trichothecenes from Fusarium sporotrichioides. Trichodiene synthase (TS) encoded by tri5 gene in Trichoderma catalyses the conversion of farnesyl pyrophosphate to trichodiene as reported earlier. In this study, we have carried out a comprehensive comparative sequence and structural analysis of the TS, which revealed the conserved residues involved in catalytic activity of the protein. In silico, modelled tertiary structure of TS protein showed stable structural behaviour during simulations. Two single-substitution mutants, i.e. D109E, D248Y and one double-substitution mutant (D109E and D248Y) of TS with potentially higher activities are screened out. The mutant proteins showed more stability than the wild type, an increased number of electrostatic interactions and better binding energies with the ligand, which further elucidates the amino acid residues involved in the reaction mechanism. These results will lead to devise strategies for higher TS activity to ultimately enhance the trichodermin production by Trichoderma spp. for its better exploitation in the sustainable agricultural practices.