Effect of deletion of a plant like pentapeptide insert on kinetic, structural and immunological properties of enolase from Plasmodium falciparum.

Plasmodium falciparum enolase (Pfen) is of photosynthetic lineage as evident from the presence of a plant like pentapeptide insert (104)EWGWS(108) in a highly conserved surface loop of the protein. Such a unique region which is absent in human enolase, constitutes an excellent target for inhibitor design, provided its essentiality for function could be demonstrated. A deletion Pfen lacking this insert was made and the effect of this deletion on activity and structure was assessed. Deletion of insert resulted in approximately 100-fold decrease in k(cat)/K(m) and caused dissociation of dimeric form into monomers. Since the parasite enolase localizes on the merozoite surface and confers partial protection against malaria [I. Pal-Bhowmick, M. Mehta, I. Coppens, S. Sharma, G.K. Jarori, Infect. Immun. 75(11) (2007) 5500-5008], the possibility of the insert being involved in protective response was examined. Serum from Pfen vaccinated mouse which showed prolonged survival to parasite challenge had negligible reactivity against deletion protein as compared to wild type enolase. These results indicate that the insert sequence is required for the full enolase activity and may constitute the protective antigenic epitope in parasite enolase.

Recreating the synthesis of starch granules in yeast.

Starch, as the major nutritional component of our staple crops and a feedstock for industry, is a vital plant product. It is composed of glucose polymers that form massive semi-crystalline granules. Its precise structure and composition determine its functionality and thus applications; however, there is no versatile model system allowing the relationships between the biosynthetic apparatus, glucan structure and properties to be explored. Here, we expressed the core Arabidopsis starch-biosynthesis pathway in Saccharomyces cerevisiae purged of its endogenous glycogen-metabolic enzymes. Systematic variation of the set of biosynthetic enzymes illustrated how each affects glucan structure and solubility. Expression of the complete set resulted in dense, insoluble granules with a starch-like semi-crystalline organization, demonstrating that this system indeed simulates starch biosynthesis. Thus, the yeast system has the potential to accelerate starch research and help create a holistic understanding of starch granule biosynthesis, providing a basis for the targeted biotechnological improvement of crops.