Properties of family 79 β-glucuronidases that hydrolyze β-glucuronosyl and 4-O-methyl-β-glucuronosyl residues of arabinogalactan-protein
Properties of family 79 β-glucuronidases that hydrolyze β-glucuronosyl and 4-O-methyl-β-glucuronosyl residues of arabinogalactan-protein
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DOI：
10.1016/j.carres.2008.03.004
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发表时间：
2008-05-19
影响因子：
3.1
通讯作者：
Tsumuraya, Yoichi
中科院分区：
文献类型：
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作者：
Konishi, Tomoyuki;Kotake, Toshihisa;Tsumuraya, Yoichi
阿拉伯半乳糖蛋白(AGPs)的碳水化合物部分是这些蛋白多糖在高等植物中发挥生理功能所必需的，它主要由Gal、L-Ara、GLCA和4-Me-GLCA残基组成。在这项研究中，我们根据从黑曲霉中提纯的天然β-葡萄糖醛酸酶的氨基酸序列，在黑曲霉和粗脉孢霉中分别鉴定了编码79β-葡萄糖醛酸酶家族的两个基因，分别命名为AnGlcAase和NcGlcAase。尽管AnGlcAase和NcGlcAase的推导蛋白序列高度相似，但在毕赤酵母中表达的重组酶对AGPs的4-Me-Glca残基表现出明显的底物特异性：重组ADGlcAase(RAnGlcAase)显著地释放了萝卜AGPs中的GLCA和4-Me-Glca残基，而重组NcGlcAase(RNcGlcAase)对AGPS的4-Me-GLCA残基的活性很低。RAnGlcAase对PNPβ-Glca的最大水解酶活性出现在pH 3.0-4.0之间，而rNcGlcAase的最大活性出现在pH 6.0。RAnGlcAase对PNPβ-Glca和β-GICA-(1-gt；6)-Gal的表观K值分别为30.4和422 mU，rNcGlcAase的表观K值分别为38.3和378 mU。与天然酶相似，rAnGlcAase能够催化PNPβ-Glca残基向各种单糖受体如GLC、Gal和Xyl的转糖基化。我们认为AnGlcAase和NcGlcAase都是一种新型的β-葡萄糖醛酸苷酶，具有降解AGP的β-Glca和4-Me-β-Glca残基的能力，尽管它们的偏好有很大的不同。(C)2008爱思唯尔有限公司。保留所有权利。
The carbohydrate moieties of arabinogalactan-proteins (AGPs), which are mainly composed of Gal, L-Ara, GlcA, and 4Me-GlcA residues, are essential for the physiological functions of these proteoglycans in higher plants. For this study, we have identified two genes encoding family 79 beta-glueuronidases, designated AnGlcAase and NcGlcAase, in Aspergillus niger and Neurospora crassa, respectively, based on the amino acid sequence of a native beta-glucuronidase purified from a commercial pectolytic enzyme preparation from A. niger. Although the deduced protein sequences of AnGlcAase and NcGlcAase were highly similar, the recombinant enzymes expressed in Pichia pastoris exhibited distinct substrate specificity toward 4-Me-GlcA residues of AGPs: recombinant ADGlcAase (rAnGlcAase) substantially liberated both GlcA and 4-Me-GlcA residues from radish AGPs, whereas recombinant NcGlcAase (rNcGlcAase) activity on the 4-Me-GlcA residues of AGPs was very low. Maximum activity of rAnGlcAase hydrolyzing PNP beta-GlcA occurred at pH 3.0-4.0, whereas the maximum rNcGlcAase activity was at pH 6.0. The apparent K. values of rAnGlcAase were 30.4 mu M for PNP beta-GlcA and 422 mu M for beta-GIcA-(1 -> 6)-Gal, and those of rNcGlcAase were 38.3 mu M and 378 mu M, respectively. Similar to the native enzyme, rAnGlcAase was able to catalyze the transglycosylation of GlcA residues from PNP beta-GlcA to various monosaccharide acceptors such as Glc, Gal, and Xyl. We propose that both AnGlcAase and NcGlcAase are instances of a novel type of beta-glucuronidase with the capacity to hydrolyze beta-GlcA and 4-Me-beta-GlcA residues of AGPs, although they differ significantly in their preferences. (c) 2008 Elsevier Ltd. All rights reserved.