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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Publications at this Location » Publication #341403
Research Project: Technologies for Improving Process Efficiencies in Biomass Refineries

Location: Bioenergy Research

Title: Absence or presence of metal ion activation in two structurally similar GH43 beta-xylosidases

Author
item Jordan, Douglas
item STOLLER, J. ROSE - Former ARS Employee
item Kibblewhite, Rena
item Lee, Charles
item Wagschal, Kurt

Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/22/2018
Publication Date: 3/24/2018
Citation: Jordan, D.B., Stoller, J.R., Kibblewhite, R.E., Lee, C.C., Wagschal, K.C. 2018. Analysis of divalent-metal requirements of two GH43 ß-xylosidases by site-directed mutagenesis. Enzyme and Microbial Technology. 114:29-32. doi: 10.1016/j.enzmictec.2018.03.007.
DOI: https://doi.org/10.1016/j.enzmictec.2018.03.007

Interpretive Summary: One of our project goals is to identify highly active enzymes that could perform well in saccharification reactors to help convert hemicelluloses to constituent monomers, which can then be fermented to bioethanol and other valuable products. In working on xylosidases, we discovered that some of these enzymes use divalent metal cofactors to catalyze their reaction. The enzymes have similar amino acid sequences, particularly in the active site. Here we report a xylosidase that also has a similar amino acid sequence, but does not use a metal cofactor in catalysis.

Technical Abstract: Two GH43 ß-xylosidases, RS223-BX and BoXA, that share 19 of 20 active-site residues were compared by using site-directed mutagenesis. RS223-BX is strongly activated by divalent-metal cations: kcat 32-fold, kcat/Km 84-fold, and the X-ray structure of this enzyme shows that a Ca2+ cation is chelated by an active-site Asp carboxyl group and an active-site His. Mutation to Ala causes 90% loss of activity for the Asp mutant and 98% for the His mutant, indicating their importance to catalysis. For the other enzyme (BoXA), mutation to Ala causes 20% loss of activity for the His mutant and 40% gain of activity for the Asp mutant, indicating the lack of importance for activity of the native residues and the lack of metal-dependency, given that the Asp residue is a much better metal chelator than His and occupies the active site to secure the metal cation in known metal ion dependent GH43 xylosidases.