Structural variations of transketolases over the evolution from bacteria to higher eukaryotes: In silico/in vitro...

Transketolases are ubiquitous enzymes that predominantly control the pentose phosphate pathway and are involved in the synthesis of aromatic amino acids, nucleotides and the regulation of oxidative stress. The development of specific inhibitors of human pathogen transketolases could provide a new class of antibiotics. To answer this question, it is necessary to compare human transketolase with those of pathogenic organisms to ensure they are sufficiently different. This chapter presents two protocols for the expression of human and of thirteen transketolases from priority pathogenic organisms, in order to obtain six new experimental structures by X-ray diffraction. Resolution of the electron density maps was performed using in silico models, using detailed protocol for their generation and validation. The experimental structures and models made possible to map and compare for the first time active sites and monomer-monomer interfaces of transketolases. Being at least 50 residues shorter, animal transketolases have evolved differently from those of bacteria, fungi and parasites. The comparison of the monomer-monomer interface also demonstrates that this zone is highly specific to each transketolase, in contrast to their conserved active site. However, in both areas, human transketolase has a significantly higher number of non-covalent bonds than pathogen transketolases, probably to maintain its shorter structure. These observations suggest that pathogen transketolases can be specifically inhibited, particularly targeting the monomer-monomer interface, without affecting human transketolase activity.