Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

Abstract

Based on sequence alignment of selected Cl − dependent and independent glycoside hydrolase family 13 enzymes, two invariant residues (Arg201 and Asn347) and one tyrosine (Tyr365) that might be responsible for the binding of Bacillus licheniformis trehalose-6-phosphate hydrolase (Bl TreA) to chloride ion were identified. The role of these three residues was further explored by mutational and biophysical analyses. The mutant enzymes (R201Q/E/K, N327Q/D/K, and Y365A/R) and Bl TreA were individually overexpressed in Escherichia coli M15 host cells and purified by one-step nickel affinity chromatography on Ni-NTA resin. The purified Bl TreA and Y365A had a specific activity of 236.9 and 47.6 U/mg protein, respectively. The remaining enzymes lost their hydrolase activity completely even in the presence of high salt. With the exception of Y365A, all mutant enzymes did not have the ability to bind fluoride, chloride and nitrate anions. Structural analyses showed that the circular dichroism spectra of the mutant proteins were consistent with those of Bl TreA. However, wild-type and mutant enzymes displayed a slight difference in the profiles of intrinsic tryptophan fluorescence. Collectively, these results clearly indicate that Arg201 and Agr327 residues might play an essential role in chloride binding of Bl TreA.

Molecular and Biochemical Characterization of Salt-Tolerant Trehalose-6-Phosphate Hydrolases Identified by Screening and Sequencing Salt-Tolerant Clones From the Metagenomic Library of the Gastrointestinal Tract

“…3 From the results, we found a site Asn327 marked by a pentangle ( Figure 3 ). The site is considered essential for binding to chloride ions according to Ong et al (2014) . Moreover, the findings may help explain why TRE_P2 and TRE_P3 confer salt tolerance.…”

“…Besides, the considerable amount of negatively charged acid residues dispersed on the outer layer of the protein may also play a part in it as it constituted a solvation case, preventing the protein layer from dehydrating and helping the protein adapt salinity (De Santi et al, 2016;Wang et al, 2016). Ong et al (2014) also found out that the efficiency of BlTreA and Y365A enzymes toward pNPG was limited when they were desalted, maintaining less than 15.8% of the TreA activity. Given this salt-tolerance property, rTRE_P2 and rTRE_P3 are useful for reporter genes (Sniezko et al, 1998) and other salttolerant environments.…”

“…According to current findings, trehalose-6-phosphate hydrolases are identified in E. coli ( Rimmele and Boos, 1994 ), Bacillus subtilis ( Sniezko et al, 1998 ), Bacillus sp. GP16 ( Karelov et al, 2003 ), Bacillus licheniformis ( Chuang et al, 2012 ; Ong et al, 2014 ; Chen et al, 2015 ; Lin et al, 2016 ), Lactobacillus acidophilus ( Duong et al, 2006 ), or Klebsiella pneumoniae ( Wu et al, 2011 ), among which Lactobacillus acidophilus is a probiotic microorganism existing in the human gastrointestinal environment. At present, the largest amount of reported trehalose-6-phosphate hydrolases found were from Bacillus .…”

“…The biochemical characterization research of trehalose-6-phosphate hydrolase mainly focused on the substrate specificity ( Rimmele and Boos, 1994 ), kinetic parameters ( Karelov et al, 2003 ), structure ( Ong et al, 2014 ), and the effect of sugar osmolytes on the refolding of trehalose-6-phosphate hydrolase ( Chen et al, 2015 ). For example, Karelov et al (2003) used T6P and p NPG as substrates to determine the K M and K cat values of trehalose-6-phosphate hydrolase at different pHs, temperatures, and NaCl conditions, respectively.…”

“… Rimmele and Boos (1994) mainly determined the substrate specificity of trehalose-6-phosphate hydrolase. Ong et al (2014) explored the effect of Arg201, Asn327, and Tyr365 on chloride ion binding of trehalose-6-phosphate hydrolase through mutation and biophysical analysis. Only trehalose-6-phosphate hydrolase BlTreA ( Chuang et al, 2012 ) from Bacillus licheniformis have been studied in detail.…”