Structural characterization of CA1462, the Candida albicans thiamine pyrophosphokinase

We report the enzymatic characterization and the crystallographic structure of the Candida albicans Thiamine pyrophosphokinase. The protein was co-crystallized with thiamine or thiamine-PNP.The presence of an inorganic phosphate in the crystallographic structure opposite the known AMP binding site relative to the thiamine moiety suggests that a second AMP molecule could be accommodated in the C. albicans structure. Together with the crystallographic structures of the enzyme/substrate complexes this suggests the existence of a secondary, less specific, nucleotide binding site in the Candida albicans thiamine pyrophosphokinase which could transiently serve during the release or the binding of ATP. The structures also highlight a conserved Glutamine residue (Q138) which could interact with the ATP α-phosphate and act as gatekeeper. Finally, the TPK/Thiamine-PNP complex is consistent with a one step mechanism of pyrophosphorylation.Our laboratory runs a structural genomics project (PROFUN [1]) targeting fungal protein-coding genes in search of new anti-fungal targets. The main goal of this project is to quantitatively express the selected Saccharomyces cerevisiae and Candida albicans genes products to characterize them functionally and structurally as well as explore their potential as new drug targets. The genes selected in Candida albicans are of two types: 1) they have orthologous genes in Saccharomyces cerevisiae that are essential or 2) they are conserved in pathogenic fungal genomes and can be absent from the non-pathogenic yeast Saccharomyces cerevisiae.Candida albicans is a diploid organism with eight sets of homologous chromosomes and a genome size of about 32 Mb. This pathogen is part of the normal microflora of the human gastrointestinal tract or oropharynx and is responsible for various nosocomial infections, potentially lethal in immunocompromised patients or patients in the intensive care unit. The CA1462 protein is encoded by a gene conserved across all k