Engineering of the catalytic site of xylose isomerase to enhance bioconversion of a non-preferential substrate

Protein Engineering, Design & Selection : PEDS
Darshan H PatelHyeun-Jong Bae

Abstract

Mutation in active site would either completely eliminate enzyme activity or may result in an active site with altered substrate-binding properties. The enzyme xylose isomerase (XI) is sterospecific for the α-pyranose and α-fructofuranose anomers and metal ions (M1 and M2) play a pivotal role in the catalytic action of this enzyme. Mutations were created at the M2 site of XI of Thermus thermophilus by replacing D254 and D256 with arginine. Mutants D254R and a double mutant (D254R/D256R) showed complete loss of activity while D256R showed an increase in the specificity on D-lyxose, L-arabinose and D-mannose which are non-preferential substrates for XI. Both wild type (WT) and D256R showed higher activity at pH 7.0 and 85°C with an increase in metal requirement. The catalytic efficiency Kcat/Km (S(-1) mM(-1)) of D256R for D-lyxose, L-arabinose and D-mannose were 0.17, 0.09 and 0.15 which are higher than WT XI of T.thermophilus. The altered catalytic activity for D256R could be explained by the possible role of arginine in catalytic reaction or the changes in a substrate orientation site. However, both the theories are only assumptions and have to be addressed with crystal study of D256R.

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Citations

Dec 23, 2015·Microbial Cell Factories·Moez RhimiEmmanuelle Maguin
Aug 19, 2015·Biomacromolecules·Sajeesh Kumar Madhurakkat PerikamanaHeungsoo Shin
Jan 16, 2013·Journal of Molecular Biology·Moshe Ben-DavidDan S Tawfik
Oct 14, 2021·Critical Reviews in Biotechnology·Renan Yuji MiyamotoLeticia Maria Zanphorlin

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