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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Publications at this Location » Publication #293692
Title: Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism ß-xylosidases

Author
item Jordan, Douglas
item Vermillion, Karl
item GRIGORESCU, ARABELA - Northwestern University
item Braker, Jay

Submitted to: Archives of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/22/2013
Publication Date: 9/15/2013
Publication URL: http://handle.nal.usda.gov/10113/62093
Citation: Jordan, D.B., Vermillion, K., Grigorescu, A.A., Braker, J.D. 2013. Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism ß-xylosidases. Archives of Biochemistry and Biophysics. 537(2):176-184.

Interpretive Summary: More efficient enzymes for breaking down cellulose and hemicellulose are needed to bring down production costs of bioethanol and other biofuels. This paper corrects some literature mistakes on an enzyme that is used to break down xylan, a major hemicellulose. Its publication will help other researchers in the field avoid the mistakes and be aware of the mistakes as they occur in the literature. In addition, the corrected data itself shows new properties that were not realized before.

Technical Abstract: We obtained Cx1 from a commercial supplier, whose catalog listed it as a ß-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein sequencing indicates Cx1 is similar to but not identical to ß- xylosidases of GH52, including Q09LZ0, that have retaining mechanisms. Q09LZ0 ß-xylosidase had been characterized biochemically in kinetic reactions that contained Tris. We overproduced Q09LZ0 and demonstrated that Tris is a competitive inhibitor of the ß-xylosidase. Also, the previous work used grossly incorrect extinction coefficients for product 4-nitrophenol. We redetermined kinetic parameters using reactions that omitted Tris and using correct extinction coefficients for 4-nitrophenol. Cx1 and Q09LZ0 ß-xylosidases were thus shown to possess similar kinetic properties when acting on 4-nitrophenyl-ß-D-xylopyranoside and xylobiose. kcat pH profiles of Cx1 and Q09LZ0 acting on 4-nitrophenyl-ß-D-xylopyranoside and xylobiose have patterns containing two rate increases with increasing acidity, not reported before for glycoside hydrolases. The dexylosylation step of 4-nitrophenyl-ß-D-xylopyranoside hydrolysis mediated by Q09LZ0 is not rate determining for kcat 4NPX.