Research Article: Purification and biochemical characterization of FrsA protein from Vibrio vulnificus as an esterase

Date Published: April 5, 2019

Publisher: Public Library of Science

Author(s): Xiaoqin Wang, Zhi-Min Li, Qingyue Li, Mingsong Shi, Lingling Bao, Dingguo Xu, Zhimin Li, Andy T. Y. Lau.

http://doi.org/10.1371/journal.pone.0215084

Abstract

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 Km and kcat 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.

Partial Text

Fermentation-respiration switch protein (FrsA) was initially identified as a novel protein binding to the unphosphorylated form of IIAGlc specifically in Escherichia coli extracts [1]. FrsA was thought to be involved in the metabolic flux between respiration and fermentation pathways by interacting with IIAGlc in E. coli [1]. Disruption of frsA gene in E. coli resulted in increased respiration rate when glucose was used as carbon source, whereas overexpression of FrsA accelerated fermentation rate on glucose with concurrent accumulation of mixed-acid fermentation products [1]. Although the physiological function of FrsA was implicated in metabolism, its biochemical function was not clear yet.

VvFrsA was biochemically identified to function as a cofactor-independent pyruvate decarboxylase with the highest activity reported so far [2]. The kcat of VvFrsA as a pyruvate decarboxylase was reported to be approximately 1400 s-1, which was considerably higher than that of TPP-dependent pyruvate decarboxylase from other species such as Saccharomyces cerevisiae [31]. This extraordinary finding, if correct, cast contradiction with current cognition that nature evolved cofactor such as TPP to stabilize the generated acyl carbanions during the oxidative and non-oxidative decarboxylation of α-keto acids in all kingdoms of life. However, this remarkable claim was disputed by Kellett et al., who demonstrated that VvFrsA was not a cofactor-independent pyruvate decarboxylase with computational, structural and kinetic evidences later [3]. With these contradictory observations in mind, we expressed the recombinant VvFrsA with E. coli expression system and purified the protein by Ni-NTA affinity chromatography.

In this study, pET28a-VvFrsA expression vector was constructed and recombinant VvFrsA protein was purified by Ni-NTA affinity chromatography. Based on the amino acid sequence homology and the crystal structure of α/β hydrolase fold, the hydrolytic activity of VvFrsA for esters was determined for the first time. Our results demonstrated that the protein was able to catalyze the hydrolysis of esters, especially p-nitrophenyl acetate, and the protein was kinetically characterized with p-nitrophenyl acetate as substrate in detail.

 

Source:

http://doi.org/10.1371/journal.pone.0215084

 

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