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PEDS Advance Access originally published online on February 15, 2005
Protein Engineering Design and Selection 2004 17(12):861-869; doi:10.1093/protein/gzh099
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions{at}oupjournals.org

Engineering the substrate specificity of xylose isomerase

Johanna Karimäki1, Tarja Parkkinen2, Harri Santa1, Ossi Pastinen1, Matti Leisola1, Juha Rouvinen2 and Ossi Turunen1,3

1Laboratory of Bioprocess Engineering, Helsinki University of Technology, PO Box 6100, 02015-HUT and 2Department of Chemistry, University of Joensuu, PO Box 111, 80101 Joensuu, Finland

3 To whom correspondence should be addressed. E-mail: ossi.turunen{at}hut.fi

Xylose isomerase (XI) catalyzes the isomerization and epimerization of hexoses, pentoses and tetroses. In order to clarify the reasons for the low reaction efficiency of a pentose sugar, L-arabinose, we determined the crystal structure of Streptomyces rubiginosus XI complexed with L-arabinose. The crystal structure revealed that, when compared with D-xylose and D-glucose, L-arabinose binds to the active site in a partially different position, in which the ligand has difficulties in binding the catalytic metal M2. Lys183 has been thought to stabilize the open substrate conformation by hydrogen bonding to oxygen O1. Our results with L-arabinose showed that the substrate stays in a linear form even without a hydrogen bond between Lys183 and oxygen O1. We engineered mutations to the active site of Actinoplanes missouriensis XI to improve the reaction efficiency with L-arabinose. The mutation F26W was intended to shift the position of oxygen O1 of L-arabinose closer to the catalytic metal M2. This effect of F26W was modeled by free energy perturbation simulations. In line with this, F26W increased 2-fold the catalytic efficiency of XI with L-arabinose; the increase was seen mainly in kcat. The mutation Q256D was outside the sphere of the catalytic residues and probably modified the electrostatic properties of the active site. It improved 3-fold the catalytic efficiency of XI with L-arabinose; this increase was seen in both Km and kcat. This study showed that it is possible to engineer the substrate specificity of XI.

Received July 2, 2004; revised January 4, 2005; accepted January 4, 2005.

Edited by Bauke Dijkstra


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