Structural studies of three enzymes: telomerase, the methyltransferase CobJ and pectate lyase

Abstract

This thesis investigates the structure and function of three enzymes of biotechnological and biomedical interest: telomerase from Caenorhabtidis elegans, pectate lyase from Bacillus subtilis and the methyltransferase CobJ from Rhodobacter capsulatus. Telomerase is a ribonucleoprotein found in all eukaryotes and its function is to maintain telomere length, sustain chromosome integrity and circumvent the end-replication problem. The protein requires two subunits to function, telomerase reverse transcriptase (TERT), the catalytic component, and an intrinsic RNA template (TR). The TR makes telomerase a unique reverse transcriptase using the template in the synthesis of short iterative sequences which cap the ends of telomeres. This work reports the successful cloning of a small and therefore potentially crystallisable TERT from C. elegans and expression trials of this catalytic component. Cobalamin (vitamin B12) is an intricate small molecule belonging to a group of compounds called cyclic tetrapyrroles. Its biosynthesis is achieved through a complex pathway encompassing over thirty different enzyme-mediated reactions. Within this pathway there are seven methyltransferases which add eight S-adenosyl-methionine (SAM) derived methyl groups to the macrocycle. CobJ catalyses the methylation of C17 and ring contraction at C20, this reaction which exudes C20 from the tetrapyrrole ring is unprecedented in nature. In this thesis I report the crystallisation of native CobJ and refinement and validation of a high resolution structure along side co-crystallisation and soaking experiments aimed at capturing an enzyme-tetrapyrrole complex. Pectate lyase (BsPel) is an enzyme secreted from the bacterium B. subtilis, it is one of many enzymes secreted by plant pathogens that is responsible for soft rot disease in plants and vegetables. The lyase utilises anti β-elimination chemistry to cleave an α-1,4- glycosidic link present in polygalacturonate the major component of the plant cell wall. 3 The structure of BsPel in complex with hexagalacturonate and a cobalt metal has been solved confirming the position and role of the putative catalytic base Arg 279 in the abstraction of a proton from C5 in galacturonate.