Crystal Structure and Functional Characterization of a Xylose Isomerase (PbXI) from the Psychrophilic Soil Microorganism, Paenibacillus sp.

Abstract

Xylose isomerase (XI; E.C.5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at 60 degrees C. We solved the crystal structure of PbXI at 1.94-angstrom resolution to investigate the origin of its thermostability. The PbXI structure shows a (beta/alpha)(8)-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the beta 4-alpha 6 loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the beta 1-alpha 2 loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible beta 1-alpha 2 loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate low-temperature purpose-specific XI enzymes.