STRUCTURAL STUDY OF THIAMINE MONOPHOSPHATE KINASE (ThiL) IN GRAM-NEGATIVE BACTERIA

The World Health Organization stated that, in the last decade, the number of deaths due to antibiotic resistance has been increasing, so the discovery of new drugs and new therapeutic targets is necessary since, at present, the existing antibiotics are limited to only five targets/pathways.
Recently, the enzyme thiamine phosphate kinase (ThiL), a kinase with a role in the phosphorylation of thiamine to its active form (vitamin B1), was evaluated as a possible therapeutic target in Pseudomonas aeruginosa. This study's objective is to provide a complete structure-function relationship of this enzyme in Gram-negative bacteria and move forward toward the discovery of potential inhibitors. Preliminary results for ThiL from Klebsiella pneumoniae, indicate an excellent solubility and yield (12 mg per culture L). DSF measurements indicate that the stability of the protein increases in the presence of magnesium in combination with substrates (thiamine monophosphate and adenosine triphosphate) and products (thiamine pyrophosphate and adenosine diphosphate). The crystal structure of the enzyme was determined at 1.52 Å resolution with synchrotron radiation (still under refinement). Structure-based comparisons of the substrate (thiamine monophosphate) with different organic compound databases were initiated in a search for possible inhibitor candidates. So far, only six organisms have the structure of their ThiL enzymes determined. This study contributes with the structural characterization of a novel ThiL and allows future structure-based campaigns for the design of new inhibitors.
This work was supported by Conselho Nacional de Desenvolvimiento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP # 2022/04298-3).