Nature-inspired Enzyme engineering and sustainable catalysis: biochemical clues from the world of plants and extremophiles.

The use of enzymes to accelerate chemical reactions for the synthesis of industrially important products is rapidly gaining popularity. Biocatalysis is an environment-friendly approach as it not only uses non-toxic, biodegradable, and renewable raw materials but also helps to reduce waste generation. In this context, enzymes from organisms living in extreme conditions (extremozymes) have been studied extensively and used in industries (food and pharmaceutical), agriculture, and molecular biology, as they are adapted to catalyze reactions withstanding harsh environmental conditions. Enzyme engineering plays a key role in integrating the structure-function insights from reference enzymes and their utilization for developing improvised catalysts. It helps to transform the enzymes to enhance their activity, stability, substrates-specificity, and substrate-versatility by suitably modifying enzyme structure, thereby creating new variants of the enzyme with improved physical and chemical properties. Here, we have illustrated the relatively less-tapped potentials of plant enzymes in general and their sub-class of extremozymes for industrial applications. Plants are exposed to a wide range of abiotic and biotic stresses due to their sessile nature, for which they have developed various mechanisms, including the production of stress-response enzymes. While extremozymes from microorganisms have been extensively studied, there are clear indications that plants and algae also produce extremophilic enzymes as their survival strategy, which may find industrial applications. Typical plant enzymes, such as ascorbate peroxidase, papain, carbonic anhydrase, glycoside hydrolases and others have been examined in this review with respect to their stress-tolerant features and further improvement via enzyme engineering. Some rare instances of plant-derived enzymes that point to greater exploration for industrial use have also been presented here. The overall implication is to utilize biochemical clues from the plant-based enzymes for robust, efficient, and substrate/reaction conditions-versatile scaffolds or reference leads for enzyme engineering.

Synergistic consortium of beneficial microorganisms in rice rhizosphere promotes host defense to blight-causing Xanthomonas oryzae pv. oryzae.

MAIN CONCLUSION: Rice plants primed with beneficial microbes Bacillus amyloliquefaciens and Aspergillus spinulosporus with biocontrol potential against Xanthomonas oryzae pv. oryzae, provided protection from disease by reprogramming host defence response under pathogen challenge. Plant-beneficial microbe interactions taking place in the rhizosphere are widely used for growth promotion and mitigation of biotic stresses in plants. The present study aims to evaluate the defense network induced by beneficial microorganisms in the rice rhizosphere, and the three-way interaction involved upon inoculation with dreadful bacteria Xanthomonas oryzae pv. oryzae (Xoo). Differential expression of defense-related enzymes, proteins, and genes in rice variety Swarna primed with a microbial consortium of Bacillus amyloliquefaciens and Aspergillus spinulosporus were quantified in the presence and absence of Xoo. The time-based expression profile alterations in leaves under the five distinct treatments "(unprimed unchallenged, unprimed Xoo challenged, B. amyloliquefaciens primed and challenged, A. spinulosporus primed and challenged, B. amyloliquefaciens and A. spinulosporus consortium primed and challenged)" revealed differential early upregulation of SOD, PAL, PO, PPO activities and TPC content in beneficial microbes primed plants in comparison to unprimed challenged plants. The enhanced defense response in all the rice plants recruited with beneficial microbe was also reflected by reduced plant mortality and an increased plant dry biomass and chlorophyll content. Also, more than 550 protein spots were observed per gel by PD Quest software, a total of 55 differentially expressed protein spots were analysed used MALDI-TOF MS, out of which 48 spots were recognized with a significant score with direct or supporting roles in stress alleviation and disease resistance. qRT-PCR was carried out to compare the biochemical and proteomic data to mRNA levels. We conclude that protein biogenesis and alleviated resistance response may contribute to improved biotic stress adaptation. These results might accelerate the functional regulation of the Xoo-receptive proteins in the presence of beneficial rhizospheric microbes and their computation as promising molecular markers for superior disease management.

Antioxidant activities of novel resveratrol analogs in breast cancer.

The objective of the present study was to characterize the role of novel resveratrol (Res) analogs: 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1, 2-diol} (HPIMBD) and 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD) as potent antioxidants against breast cancer. Non-neoplastic breast epithelial cell lines MCF-10A and MCF-10F were treated with 17ß-estradiol (E2), Res, HPIMBD, and TIMBD for up to 72 h. mRNA and protein levels of antioxidant genes, superoxide dismutase 3 (SOD3) and N-quinoneoxidoreductase-1 (NQO1) and transcription factors, nuclear factor erythroid 2-related factor (Nrf) 1, 2 and 3 were quantified after the above treatments. Generation of reactive oxygen species (ROS) was measured by CM-H2-DCFDA and oxidative-DNA damage was determined by measuring 8-hydroxy-2-deoxyguanosine (8-OHdG). HPIMBD and TIMBD scavenged cellular ROS production, attenuated oxidative DNA damage, increased mRNA and protein expression levels of SOD3 and NQO1 and activated Nrf signaling pathway. Our studies demonstrate that HPIMBD and TIMBD have the potential as novel antioxidants to prevent development of breast cancer.

Tamoxifen synergizes with 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} and 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, novel azaresveratrol analogs, in inhibiting the proliferation of breast cancer cells.

We have recently shown that 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} (HPIMBD) and 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD), novel analogs of resveratrol (Res), selectively inhibited the proliferation of breast cancer cells. In the current study, we tested HPIMBD and TIMBD individually in combination with tamoxifen (Tam) for inhibition of growth of breast cancer cells. Tamoxifen was first tested on non-neoplastic breast epithelial cell lines and its dose that does not inhibit their growth was determined. A combination of this low dose of Tam with either of the Res analogs HPIMBD or TIMBD, resulted in synergistic inhibition of proliferation of breast cancer cells. Both estrogen receptor (ER)-positive and negative breast cancer cell lines responded to the combination. The combination resulted in a substantial decrease in IC50 values of Res analogs in all breast cancer cell lines tested. Mechanistic studies showed a synergistic increase in apoptosis and autophagy genes (beclin-1 and LC3BII/I) with the combination in ER-negative MDA-MB-231 cells. In ER-positive MCF-7 and T47D cells, the mechanism of synergy was found to be inhibition of expression of ERα and oncogene c-Myc. The combination treatment had a synergistic effect in inhibiting the colony forming and spheroid forming ability of cancer cells. Taken together, our findings indicate that a combination of Tam and Res analogs HPIMBD or TIMBD represents a novel approach to enhancing the use of Tam in therapy for breast cancers. Considering the urgent need for novel therapeutic strategies to treat ER-negative breast cancers and overcoming resistance in ER-positive cancers, this combinatorial approach is worthy of continued investigation.

4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, 1 a novel resveratrol analog, differentially regulates estrogen receptors α and ß in breast cancer cells.

Breast cancer is a public health concern worldwide. Prolonged exposure to estrogens has been implicated in the development of breast neoplasms. Epidemiologic and experimental evidence suggest a chemopreventive role of phytoestrogens in breast cancers. Resveratrol, a naturally occurring phytoestrogen, has been shown to have potent anti-cancer properties. However, poor efficacy and bioavailability have prevented the use of resveratrol in clinics. In order to address these problems, we have synthesized a combinatorial library of azaresveratrol analogs and tested them for their ability to inhibit the proliferation of breast cancer cells. We have recently shown that 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD), has better anti-cancer properties than resveratrol and any other resveratrol analog we have synthesized so far. The objective of this study was to investigate the regulation of estrogen receptors (ERs) α and ß by TIMBD in breast cancer cell lines. We demonstrate that TIMBD significantly induces the mRNA and protein expression levels of ERß and inhibits that of ERα. TIMBD inhibits mRNA and protein expression levels of oncogene c-Myc, and cell cycle protein cyclin D1, which are important regulators of cellular proliferation. TIMBD significantly induces protein expression levels of tumor suppressor genes p53 and p21 in MCF-7 cells. TIMBD inhibits c-Myc in an ERß-dependent fashion in MCF-10A and ERß1-transfected MDA-MB-231 cells, suggesting regulation of ERs as an important upstream mechanism of this analog. ERß plays a partial role in inhibition of proliferation by TIMBD while ERα overexpression does not significantly affect TIMBD's inhibition.

Differential regulation of estrogen receptors α and ß by 4-(E)-{(4-hydroxyphenylimino)-methylbenzene,1,2-diol}, a novel resveratrol analog.

Breast cancer is the second leading cause of death among women in the United States. Estrogens have been implicated as major risk factors in the development of breast neoplasms. Recent epidemiologic studies have suggested a protective role of phytoestrogens in prevention of breast and other cancers. Resveratrol, a naturally occurring phytoestrogen found notably in red grapes, berries and peanuts, has been shown to possess potent anti-cancer properties. However, the poor efficacy of resveratrol has prevented its use in a clinical setting. In order to improve the efficacy of resveratrol, we have synthesized a small combinatorial library of azaresveratrol analogs and tested them for their ability to inhibit the growth of breast cancer cell lines. We have recently shown that one of the synthesized analogs, 4-(E)-{(4-hydroxyphenylimino)-methylbenzene,1,2-diol} (HPIMBD), has better anti-cancer properties than resveratrol. The objective of this study was to investigate the differential regulation of estrogen receptors (ERs) α and ß as a potential mechanism of inhibition of breast cancer by HPIMBD. Estrogen receptors α and ß have been shown to have opposing roles in cellular proliferation. Estrogen receptor α mediates the proliferative responses of estrogens while ERß plays an anti-proliferative and pro-apoptotic role. We demonstrate that HPIMBD significantly induces the expression of ERß and inhibits the expression of ERα. HPIMBD also inhibits the protein expression levels of oncogene c-Myc and cell cycle protein cyclin D1, genes downstream to ERα and important regulators of cell cycle, and cellular proliferation. HPIMBD significantly induces protein expression levels of tumor suppressors p53 and p21 in MCF-7 cells. Additionally, HPIMBD inhibits c-Myc in an ERß-dependent fashion in MCF-10A and ERß1-transfected MDA-MB-231 cells, suggesting regulation of ERs as an important upstream mechanism of this novel compound. Molecular docking studies confirm higher affinity for binding of HPIMBD in the ERß cavity. Thus, HPIMBD, a novel azaresveratrol analog may inhibit the proliferation of breast cancer cells by differentially modulating the expressions of ERs α and ß.

Natural antioxidants exhibit chemopreventive characteristics through the regulation of CNC b-Zip transcription factors in estrogen-induced breast carcinogenesis.

The objective of the present study was to characterize the role of resveratrol (Res) and vitamin C (VC) in prevention of estrogen-induced breast cancer through regulation of cap "n"collar (CNC) b-zip transcription factors. Human breast epithelial cell line MCF-10A was treated with 17ß-estradiol (E2) and VC or Res with or without E2. mRNA and protein expression levels of CNC b-zip transcription factors nuclear factor erythroid 2-related factor 1 (Nrf1), nuclear factor erythroid 2 related factor 2 (Nrf2), nuclear factor erythroid 2 related factor 3 (Nrf3), and Nrf2-regulated antioxidant enzymes superoxide dismutase 3 (SOD3) and NAD(P)H: quinone oxidoreductase 1 (NQO1) were quantified. The treatment with E2 suppressed, whereas VC and Res prevented E2-mediated decrease in the expression levels of SOD3, NQO1, Nrf2 mRNA, and protein in MCF-10A cells. The treatment with E2, Res, or VC significantly increased mRNA and protein expression levels of Nrf1. 17ß-Estradiol treatment significantly increased but VC or Res decreased Nrf3 mRNA and protein expression levels. Our studies demonstrate that estrogen-induced breast cancer might be prevented through upregulation of antioxidant enzymes via Nrf-dependent pathways.

Resveratrol inhibits estrogen-induced breast carcinogenesis through induction of NRF2-mediated protective pathways.

The importance of estrogens in the etiology of breast cancer is widely recognized. Estrogen-induced oxidative stress has been implicated in this carcinogenic process. Resveratrol (Res), a natural antioxidant phytoestrogen has chemopreventive effects against a variety of illnesses including cancer. The objective of the present study was to characterize the mechanism(s) of Res-mediated protection against estrogen-induced breast carcinogenesis. Female August Copenhagen Irish rats were treated with 17ß-estradiol (E2), Res and Res + E2 for 8 months. Cotreatment of rats with Res and E2 inhibited E2-mediated proliferative changes in mammary tissues and significantly increased tumor latency and reduced E2-induced breast tumor development. Resveratrol treatment alone or in combination with E2 significantly upregulated expression of nuclear factor erythroid 2-related factor 2 (NRF2) in mammary tissues. Expression of NRF2-regulated antioxidant genes NQO1, SOD3 and OGG1 that are involved in protection against oxidative DNA damage was increased in Res- and Res + E2-treated mammary tissues. Resveratrol also prevented E2-mediated inhibition of detoxification genes AOX1 and FMO1. Inhibition of E2-mediated alterations in NRF2 promoter methylation and expression of NRF2 targeting miR-93 after Res treatment indicated Res-mediated epigenetic regulation of NRF2 during E2-induced breast carcinogenesis. Resveratrol treatment also induced apoptosis and inhibited E2-mediated increase in DNA damage in mammary tissues. Increased apoptosis and decreased DNA damage, cell migration, colony and mammosphere formation in Res- and Res + E2-treated MCF-10A cells suggested a protective role of Res against E2-induced mammary carcinogenesis. Small-interfering RNA-mediated silencing of NRF2 inhibited Res-mediated preventive effects on the colony and mammosphere formation. Taken together, these results suggest that Res inhibits E2-induced breast carcinogenesis via induction of NRF2-mediated protective pathways.

Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated with inhibition of oxidative DNA damage in estrogen-induced breast cancer.

BACKGROUND: Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants, vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17ß-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis. METHODS: Female ACI rats were treated with E2; Vit C; Vit C + E2; BHA; and BHA + E2 for up to 240 days. mRNA and protein levels of a DNA repair enzyme 8-Oxoguanine DNA glycosylase (OGG1) and a transcription factor NRF2 were quantified in the mammary and mammary tumor tissues of rats after treatment with E2 and compared with that of rats treated with antioxidants either alone or in combination with E2. RESULTS: The expression of OGG1 was suppressed in mammary tissues and in mammary tumors of rats treated with E2. Expression of NRF2 was also significantly suppressed in E2-treated mammary tissues and in mammary tumors. Vitamin C or BHA treatment prevented E2-mediated decrease in OGG1 and NRF2 levels in the mammary tissues. Chromatin immunoprecipitation analysis confirmed that antioxidant-mediated induction of OGG1 was through increased direct binding of NRF2 to the promoter region of OGG1. Studies using silencer RNA confirmed the role of OGG1 in inhibition of oxidative DNA damage. CONCLUSIONS: Our studies suggest that antioxidants Vit C and BHA provide protection against oxidative DNA damage and E2-induced mammary carcinogenesis, at least in part, through NRF2-mediated induction of OGG1.

MicroRNA-93 regulates NRF2 expression and is associated with breast carcinogenesis.

MicroRNAs (miRNA) are small non-coding RNAs that regulate the expression of approximately 60% of all human genes and play important roles in disease processes. Recent studies have demonstrated a link between dysregulated expression of miRNAs and breast carcinogenesis. Long-term estrogen exposure is implicated in development of human breast cancers, yet underlying mechanisms remain elusive. We have recently demonstrated that antioxidant vitamin C (vit C) prevents estrogen-induced breast tumor development. In this study, we investigated the role of vit C in the regulation of microRNA-93 (miR-93) and its target gene(s) in a rat model of mammary carcinogenesis. Female August Copenhagen Irish (ACI) rats were treated with vit C in the presence or absence of 17ß-estradiol (E2) for 8 months. We demonstrate an increased expression of the miR-93 in E2-treated mammary tissues and in human breast cell lines and vit C treatment reverted E2-mediated increase in miR-93 levels. MiRNA target prediction programs suggest one of the target genes of miR-93 to be nuclear factor erythroid 2-related factor 2 (NRF2). In contrast with miR-93 expression, NRF2 protein expression was significantly decreased in E2-treated mammary tissues, mammary tumors, and in breast cancer cell lines, and its expression was significantly increased after vit C treatment. Ectopic expression of miR-93 decreased protein expression of NRF2 and NRF2-regulated genes. Furthermore, miR-93 decreased apoptosis, increased colony formation, mammosphere formation, cell migration and DNA damage in breast epithelial cells, whereas silencing of miR-93 in these cells inhibited these carcinogenic processes. Taken together, our findings suggest an oncogenic potential of miR-93 during E2-induced breast carcinogenesis.

Novel Aza-resveratrol analogs: synthesis, characterization and anticancer activity against breast cancer cell lines.

Novel Aza-resveratrol analogs were synthesized, structurally characterized and evaluated for cytotoxic activity against MDA-MB-231 and T47D breast cancer cell lines, which exhibited superior inhibitory activity than parent resveratrol compound. The binding mechanism of these compounds with estrogen receptor-α was rationalized by molecular docking studies which indicated additional hydrogen binding interactions and tight binding in the protein cavity. Induction of Beclin-1 protein expression in breast cancer cell lines after treatment with newly synthesized resveratrol analogs indicated inhibition of growth of these cell lines through autophagy. The study highlighted the advantage of introducing the imino-linkage in resveratrol motif in enhancing the anticancer potential of resveratrol suggesting that these analogs can serve as better therapeutic agents against breast cancer and can provide starting point for building more potent analogs in future.