A Rice Family 9 Glycoside Hydrolase Isozyme with Broad Substrate Specificity for Hemicelluloses in Type II Cell Walls

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Abstract
An auxin analog, 2,4-D, stimulates the activity of endo-1,4-β-glucanase (EGase) in rice ( Oryza sativa L.). The auxin-induced activity from three protein fractions was purified to homogeneity from primary root tissues (based on SDS–PAGE and isoelectric focusing after Coomassie brilliant blue staining). Amino acid sequencing indicated that the 20 N-terminal amino acid sequence of the three proteins was identical, suggesting that these proteins may be cognates of one EGase gene. An internal amino acid sequence of the the rice EGase (LVGGYYDAGDNVK) revealed that this enzyme belongs to glycosyl hydrolase family 9 (GHF9). The major isoform of this rice GHF9 [molecular weight based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS): 51,216, isoelectric point (pI): 5.5] specifically hydrolyzed 1,4-β-glycosyl linkages of carboxymethyl (CM)-cellulose, phosphoric acid-swollen cellulose, 1,3-1,4-β-glucan, arabinoxylan, xylan, glucomannan, cellooligosaccharides [with a degree of polymerization (DP) >3] and 1,4-β-xylohexaose, indicating a broader substrate range compared with those of other characterized GHF9 enzymes or EGases from higher plants. Hydrolytic products of two major hemicellulosic polysaccharides in type II cell walls treated with the purified enzyme were profiled using high-performance anion exchange chromatography (HPAEC). The results suggested that endolytic attack by rice EGase is not restricted to either the cellulose-like domain of 1,3-1,4-β-glucan or the unsubstituted 1,4-β-xylosyl backbone of arabinoxylan, but results in the release of smaller oligosaccharides (DP <6) from graminaceous hemicelluloses. The comparatively broader substrate range of this EGase with respect to β-1,4-glycan backbones (glucose and xylose) may partly reflect different roles of gramineous and non-gramineous GHF9 enzymes.