4--Methyl Modifications of Glucuronic Acids in Xylans Are Indispensable for Substrate Discrimination by GH67 α-Glucuronidase from C-125.

A GH67 α-glucuronidase gene derived from C-125 was expressed in to obtain a recombinant enzyme (GlcA67). Using the purified enzyme, the enzymatic properties and substrate specificities of the enzyme were investigated. GlcA67 showed maximum activity at pH 5.4 and 45 °C. When GlcA67 was incubated with birchwood, oat spelts, and cotton seed xylan, the enzyme did not release any glucuronic acid or 4--methyl-glucuronic acid from these substrates. GlcA67 acted only on 4--methyl-α-D-glucuronopyranosyl-(1→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (MeGlcAXyl), which has a glucuronic acid side chain with a 4--methyl group located at its non-reducing end, but did not on β-D-xylopyranosyl-(1→4)-[4--methyl-α-D-glucuronopyranosyl-(l→2)]-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylop- yranose (MeGlcAXyl) and α-D-glucuronopyranosyl-(l→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (GlcAXyl). The environment for recognizing the 4--methyl group of glucuronic acid was observed in all the crystal structures of reported GH67 glucuronidases, and the amino acids for discriminating the 4--methyl group of glucuronic acid were widely conserved in the primary sequences of the GH67 family, suggesting that the 4--methyl group is critical for the activities of the GH67 family.