IN VIVO AND IN VITRO CHARACTERIZATION OF BACTERIAL BIOTIN TRANSPORTER YIGM

• Main Author: Sophi Damayanti, Yasmine
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/49345
• Institution: Institut Teknologi Bandung

Vitamins are a heterogenous group of organic compounds required in a small amount of various biological processes. Although vitamins are essential for all living cells, they can only be synthesized de novo by microorganisms, plants, and some fungi. Vitamin uptake from environment is more favorable because the synthesis of vitamin is energetically expensive. For instance, the synthesis of one molecule of biotin (vitamin B7) in prokaryotes requires at least 6 enzymes and 7 equivalents of ATP. The ability of biotin transport in Escherichia coli has been known for several years even though E. coli does not encode BioMNY. BioMNY the most characterized biotin transporter in prokaryotes. Genomic analysis showed that yigM is a potential gene encoding biotin transporter in E. coli. YigM consists of 299 amino acids with a molecular weight of ~33 kDa. YigM is not related to ABC transporters and has no sequence similarity with any other biotin transporters. The only reported domain of YigM is a domain of unknown function (DUF6) and belongs to carboxylate/amino acid/amine/transporters (CAAT). 71% amino acids of YigM are predicted to be part of 10 transmembrane helices with the N- and C-termini in the cytoplasm. In this research, we cloned yigM from E. coli BL21(DE3) with additional sequences encoding His8×Tag and HRV 3C cleavage site at the N- or C-terminus of the protein. Growth assay in E. coli ?bioH ?yigM ?sbmA (E. coli strain with knockout in genes encoding protein that involved in biotin synthesis and transporter) was performed to determine the ability of biotin transport in vivo. The protein was solubilized in lauryl maltose neopentyl glycol (LMNG) then purified using immobilized affinity chromatography (IMAC) and size exclusion chromatography (SEC). Growth assay in E. coli ?bioH ?yigM ?sbmA showed that YigM with or without additional tag able to transport biotin. YigM was overexpressed in E. coli BL21(DE3) under the optimum condition for expression using with 0.4 mM of IPTG at 25 °C for 3 hours (in LB-medium) or 4 hours (in M9 minimal medium without biotin). However, YigM in detergent is potential to form dimer showed by a protein band of ~70 kDa in SDS-PAGE and western blot after solubilization. This probable dimer formation can be due to unspecific intermolecular interaction. Isothermal titration calorimetry (ITC) and fluorescence titration showed that apoYigM in detergent is not able to bind biotin. This could be because YigM expressed iv in E. coli BL21 (DE3) is already saturated with biotin since the strain can synthesize biotin. This possibility implies that YigM might be a high affinity biotin transporter. The other possibility is that the structure of YigM in detergent does not support biotin binding or YigM perhaps requires another protein to bind and transport biotin.