MUTAGENESIS OF RESIDUES IN SUBSTRATE BINDING POCKET OF THIT, THE S-COMPONENT OF THE ECF TRANSPORTER FOR THIAMINE

• Main Author: Aisyah
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32325
• Institution: Institut Teknologi Bandung

Thiamine pyrophosphate (TPP) is a coenzyme involved in metabolism; therefore it is essential for whole organisms. It can be synthesized from its precursors, thiamine and thiamine monophoshate (TMP), but organisms that do not have biosynthesis routes for these molecules, need transport proteins to take them up from the environment. One of the transporter proteins for thiamine is ECF ThiT, an energy-coupling factor (ECF) transporter from Lactococcus lactis. This transporter contains a substrate-binding protein called ThiT. Previous research on this protein has revealed that it has very high affinity for thiamine (KD value in picomolar range) and the thiamine-bound crystal structure has been solved. This crystal structure together with additional fluorescence experiment of thiamine analogues showed that interactions between ThiT and the positively charged nitrogen in thiamine have a major contribution to substrate binding. In this study, we elaborated the role of two residues in the substrate binding pocket of ThiT which interact with positively charged nitrogen of thiamine, Glu84 and His125. These residues have been mutated successfully into Glu84Asp, Glu84Gln, Glu84Ala, His125Phe, His125Asn, and His125Ala. To study the contribution of the functional groups of those residues, substrate binding using an intrinsic fluorescence based assay has been performed at a pH range of pH 4.0 to pH 8.0. Intrinsic fluorescence assay showed the same results for all mutant in all pH range, with thiamine affinity at least 10,000 fold lower compared to the affinity of wild type ThiT. Further experiments proved that those results were unreliable since the fluorescence quenching in those cases did not happen as an effect of thiamine binding instead as an inner filter effect from high concentration of thiamine. Isothermal titration calorimetry assay was used as more sensitive method to study the thiamine binding assay. From ITC measurement, the Glu84Ala mutant showed lower affinity more than 1,000 fold compared to that of wild type ThiT. It could be happened due to disruption of electrostatic interaction and hydrogen bonds as an effect of mutation into alanine. ITC measurement of the Glu84Gln mutant showed KD value one order of magnitude higher than that of wild type ThiT. It could be happened due to disruption of electrostatic interaction in residue 84 as an effect of neutral side chain in glutamine residue. These results suggesting that the negative charge of glutamate within the substrate binding pocket is essential for binding. The importance of the negative charge is also confirmed by the observation that the affinity of wild type ThiT at pH 4.0, which is below the pKa value of the glutamate side chain (pKa glutamate = 4.1) is decreased by 10 fold compared to pH 5.0–8.0. From these experiments, we can conclude that electrostatic interaction between negative charge of Glu84 and positive charge nitrogen from thiamine is essential for the binding. In other hand, ITC measurement of the Glu84Asp mutant showed KD value one order of magnitude higher than wild type ThiT. This result suggesting that distance of electrostatic interaction and hydrogen bond from Glu84 residue are fixed. Modification of those distances disrupts thiamine binding.