Open AccessPurification and biochemical characterization of FrsA protein from Vibrio vulnificus as an esterase
Author(s) -
Xiaoqin Wang,
Zhimin Li,
Qingyue Li,
Mingsong Shi,
Lingling Bao,
Dingguo Xu
Publication year - 2019
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0215084
Subject(s) - vibrio vulnificus , enzyme kinetics , esterase , hydrolysis , fermentation , biochemistry , protein subunit , enzyme , chemistry , substrate (aquarium) , biology , bacteria , active site , gene , ecology , genetics
Fermentation-respiration switch protein (FrsA) was thought to play an important role in controlling the metabolic flux between respiration and fermentation pathways, whereas the biochemical function of FrsA was unclear yet. A gene coding for FrsA protein from Vibrio vulnificus was chemically synthesized. The recombinant VvFrsA was expressed as a soluble protein and purified by Ni-NTA affinity chromatography. The protein had a subunit molecular weight of ca . 45 kDa by SDS-PAGE and preferred short-chain esters when p-nitrophenyl alkanoate esters were used as substrates. Optimum condition for VvFrsA was found to be at pH 9.0 and 50 °C. The protein retained high esterase activity at alkaline condition and would denature slowly at over 50 °C. With p-nitrophenyl acetate as the substrate, the K m and k cat were determined to be 18.6 mM and 0.67 s -1 , respectively, by steady-state kinetic assay. Molecular dynamics simulation and docking model structure revealed that p-nitrophenyl acetate could be the substrate of VvFrsA. In conclusion our results demonstrated that the protein was able to catalyze the hydrolysis of esters, especially p-nitrophenyl acetate, for the first time.