Crystal structure of dimeric FabZ of Plasmodium falciparum reveals conformational switching to active hexamers by peptide flips

The crystal structure of beta-hydroxyacyl acyl carrier protein dehydratase of Plasmodium falciparum (PfFabZ) has been determined at a resolution of 2.4 A. PfFabZ has been found to exist as a homodimer (d-PfFabZ) in the crystals of the present study in contrast to the reported hexameric form (h-PfFabZ) which is a trimer of dimers crystallized in a different condition. The catalytic sites of this enzyme are located in deep narrow tunnel-shaped pockets formed at the dimer interface. A histidine residue from one subunit of the dimer and a glutamate residue from the other subunit lining the tunnel form the catalytic dyad in the reported crystal structures. While the position of glutamate remains unaltered in the crystal structure of d-PfFabZ compared to that in h-PfFabZ, the histidine residue takes up an entirely different conformation and moves away from the tunnel leading to a His-Phe cis-trans peptide flip at the histidine residue. In addition, a loop in the vicinity has been observed to undergo a similar flip at a Tyr-Pro peptide bond. These alterations not only prevent the formation of a hexamer but also distort the active site geometry resulting in a dimeric form of FabZ that is incapable of substrate binding. The dimeric state and an altered catalytic site architecture make d-PfFabZ distinctly different from the FabZ structures described so far. Dynamic light scattering and size exclusion chromatographic studies clearly indicate a pH-related switching of the dimers to active hexamers.     

MUC1 oncoprotein blocks nuclear targeting of c-Abl in the apoptotic response to DNA damage

The nonreceptor c-Abl tyrosine kinase binds to cytosolic 14-3-3 proteins and is targeted to the nucleus in the apoptotic response to DNA damage. The MUC1 oncoprotein is overexpressed by most human carcinomas and blocks the induction of apoptosis by genotoxic agents. Using human carcinoma cells with gain and loss of MUC1 function, we show that nuclear targeting of c-Abl by DNA damage is abrogated by a MUC1-dependent mechanism. The results demonstrate that c-Abl phosphorylates MUC1 on Tyr-60 and forms a complex with MUC1 by binding of the c-Abl SH2 domain to the pTyr-60 site. Binding of MUC1 to c-Abl attenuates phosphorylation of c-Abl on Thr-735 and the interaction between c-Abl and cytosolic 14-3-3. We also show that expression of MUC1 with a mutation at Tyr-60 (i) disrupts the interaction between MUC1 and c-Abl, (ii) relieves the MUC1-induced block of c-Abl phosphorylation on Thr-735 and binding to 14-3-3, and (iii) attenuates the MUC1 antiapoptotic function. These findings indicate that MUC1 sequesters c-Abl in the cytoplasm and thereby inhibits apoptosis in the response to genotoxic anticancer agents.     

Development of a mutant of Trichoderma citrinoviride for enhanced production of cellulases

Considering importance of a microbial strain capable of increased cellulases production and insensitive to catabolite repression for industrial use, we have developed a mutant strain of Trichoderma citrinoviride by multiple exposures to EMS and ethidium bromide. The mutant produced 0.63, 3.12, 8.22 and 1.94 IU ml(-1) FPase, endoglucanase, beta-glucosidase and cellobiase, respectively. These levels were, respectively, 2.14, 2.10, 4.09 and 1.73 fold higher than those in parent strain. Glucose (upto 20 mM) did not repress enzyme production by the mutant under submerged fermentation conditions. In vitro activity assay with partially purified cellulase showed lack of inhibition by glucose. Interestingly, the partially purified endoglucanase and beta-glucosidase were activated by 2.0 fold and 2.6 fold, respectively, by 20 mM and 30 mM ethanol in the assay mixture. Genetic distinction of the mutant was revealed by the presence of two unique amplicans in comparative DNA fingerprinting performed using 20 random primers.     

Functional Assembly of Minicellulosomes on the Saccharomyces cerevisiae Cell Surface for Cellulose Hydrolysis and Ethanol Production

We demonstrated the functional display of a miniscaffoldin on the Saccharomyces cerevisiae cell surface consisting of three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f). Incubation with Escherichia coli lysates containing an endoglucanase (CelA) fused with a dockerin domain from C. thermocellum (At), an exoglucanase (CelE) from C. cellulolyticum fused with a dockerin domain from the same species (Ec), and an endoglucanase (CelG) from C. cellulolyticum fused with a dockerin domain from R. flavefaciens (Gf) resulted in the assembly of a functional minicellulosome on the yeast cell surface. The displayed minicellulosome retained the synergistic effect for cellulose hydrolysis. When a β-glucosidase (BglA) from C. thermocellum tagged with the dockerin from R. flavefaciens was used in place of Gf, cells displaying the new minicellulosome exhibited significantly enhanced glucose liberation and produced ethanol directly from phosphoric acid-swollen cellulose. The final ethanol concentration of 3.5 g/liter was 2.6-fold higher than that obtained by using the same amounts of added purified cellulases. The overall yield was 0.49 g of ethanol produced per g of carbohydrate consumed, which corresponds to 95% of the theoretical value. This result confirms that simultaneous and synergistic saccharification and fermentation of cellulose to ethanol can be efficiently accomplished with a yeast strain displaying a functional minicellulosome containing all three required cellulolytic enzymes.Production of bioethanol from biomass has recently attracted attention due to the mandate for a billion gallons of renewable fuel by the new Energy Policy Act (22). Current production processes using sugar cane and cornstarch are well established (19, 23). However, utilization of a cheaper substrate would render bioethanol more competitive with fossil fuel (29). Cellulosic biomass found in many low-value agricultural or wood pulping wastes is particularly well suited because of its large-scale availability, low cost, and environmentally benign production (23). The primary obstacle impeding the more widespread production of ethanol from cellulose is the absence of a low-cost technology for overcoming its recalcitrant nature (21).Recently, a new method known as consolidated bioprocessing (CBP) has been proposed that combines enzyme production, cellulose saccharification, and fermentation into a single process to dramatically reduce the cost of ethanol production (22). An ideal microorganism for CBP should possess the capability of simultaneous cellulose saccharification and ethanol fermentation. One attractive candidate is Saccharomyces cerevisiae, which is widely used for industrial ethanol production due to its high ethanol productivity and high inherent ethanol tolerance (24). Attempts have been made to engineer S. cerevisiae to hydrolyze cellulose (6, 7, 16). However, due to energetic limitations under anaerobic conditions, only a small amount of cellulases can often be secreted. An alternative is to display the cellulolytic enzymes on the yeast cell surface (13, 14). Up to three different cellulases have been displayed, permitting the hydrolysis of cellulose with concomitant ethanol production. While these results point to a potential strategy of combining ethanol-producing capability with cellulose hydrolysis, the efficiency of hydrolysis must be significantly improved before it can be employed for practical applications.Many anaerobic bacteria have developed an elaborately structured enzyme complex on the cell surface, called the cellulosome, to maximize the catalytic efficiency of cellulose hydrolysis with only a limited amount of enzymes (1, 8, 9). The major component of these cellulosome complexes is a structural scaffoldin consisting of at least one cellulose-binding domain (CBD) and repeating cohesin domains, which are docked individually with a different cellulase tagged with the corresponding dockerin domain (26). Since the interaction between dockerin and cohesin is species specific (17, 25), designer minicellulosomes composed of three different dockerin-cohesin pairs with a cellulose hydrolysis efficiency up to sixfold higher than that of similar free enzymes have been generated (11, 12). Recently, it has been shown that the specific cellulose hydrolysis rates of metabolically active cultures of C. thermocellum displaying cellulosomes are more than fourfold higher than those of purified cellulosomes (20). This significant improvement appears to be a surface phenomenon involving adhesion to cellulose for enhanced substrate capture.In the present report, we demonstrate the functional assembly of a minicellulosome composed of three different cellulases on the S. cerevisiae cell surface and the feasibility of using the engineered yeast strains for cellulosic ethanol production. The success of displaying a functional cellulosome on the surface of an organism that already produces high titers of ethanol could lay a foundation for the achievement of an industrially relevant CBP-enabling microorganism.     

Delineating the structural,functional and evolutionary relationships of sucrose phosphate synthase gene family II in wheat and related grasses

Background  

Sucrose phosphate synthase (SPS) is an important component of the plant sucrose biosynthesis pathway. In the monocotyledonous Poaceae, five SPS genes have been identified. Here we present a detailed analysis of the wheat SPSII family in wheat. A set of homoeologue-specific primers was developed in order to permit both the detection of sequence variation, and the dissection of the individual contribution of each homoeologue to the global expression of SPSII.     

Stability Performance of Inductively Coupled Plasma Mass Spectrometry-Phenotyped Kernel Minerals Concentration and Grain Yield in Maize in Different Agro-Climatic Zones

Deficiency of iron and zinc causes micronutrient malnutrition or hidden hunger, which severely affects ~25% of global population. Genetic biofortification of maize has emerged as cost effective and sustainable approach in addressing malnourishment of iron and zinc deficiency. Therefore, understanding the genetic variation and stability of kernel micronutrients and grain yield of the maize inbreds is a prerequisite in breeding micronutrient-rich high yielding hybrids to alleviate micronutrient malnutrition. We report here, the genetic variability and stability of the kernel micronutrients concentration and grain yield in a set of 50 maize inbred panel selected from the national and the international centres that were raised at six different maize growing regions of India. Phenotyping of kernels using inductively coupled plasma mass spectrometry (ICP-MS) revealed considerable variability for kernel minerals concentration (iron: 18.88 to 47.65 mg kg^–1; zinc: 5.41 to 30.85 mg kg^–1; manganese: 3.30 to17.73 mg kg^–1; copper: 0.53 to 5.48 mg kg^–1) and grain yield (826.6 to 5413 kg ha^–1). Significant positive correlation was observed between kernel iron and zinc within (r = 0.37 to r = 0.52, p < 0.05) and across locations (r = 0.44, p < 0.01). Variance components of the additive main effects and multiplicative interactions (AMMI) model showed significant genotype and genotype × environment interaction for kernel minerals concentration and grain yield. Most of the variation was contributed by genotype main effect for kernel iron (39.6%), manganese (41.34%) and copper (41.12%), and environment main effects for both kernel zinc (40.5%) and grain yield (37.0%). Genotype main effect plus genotype-by-environment interaction (GGE) biplot identified several mega environments for kernel minerals and grain yield. Comparison of stability parameters revealed AMMI stability value (ASV) as the better representative of the AMMI stability parameters. Dynamic stability parameter GGE distance (GGED) showed strong and positive correlation with both mean kernel concentrations and grain yield. Inbreds (CM-501, SKV-775, HUZM-185) identified from the present investigation will be useful in developing micronutrient-rich as well as stable maize hybrids without compromising grain yield.