Canam Thomas, Unda Faride, Mansfield Shawn D
Department of Wood Science, University of British Columbia, Vancouver, BC, Canada.
Planta. 2008 Nov;228(6):1011-9. doi: 10.1007/s00425-008-0801-6. Epub 2008 Aug 13.
The expression of two hybrid poplar cell-wall invertases (EC 3.2.1.26; PaxgINV1 and PaxgINV2) were previously shown to be spatially and temporally regulated in the vegetative tissues. The expression of PaxgINV1 was linked to processes relating to dormancy, while PaxgINV2 expression was prominent in tissues undergoing growth and expansion. In an effort to further elucidate the physiological roles of these key cell wall enzymes, PaxgINV1 and PaxgINV2 were heterologously expressed in the methylotrophic yeast Pichia pastoris. Three-dimensional predictive models of the poplar invertases revealed a structural channel containing both the conserved beta-fructofuranosidase and cell-wall invertase motifs, suggesting that this channel is the putative active site of these enzymes. Recombinant PaxgINV1 and PaxgINV2 had pH optima of 4.8 and 5.6 and temperature optima of 45 and 40 degrees C, respectively. Functional characterization revealed the ability for both enzymes to hydrolyze the fructose residue of sucrose, raffinose, stachyose and verbascose, with PaxgINV2 having higher specific activity for each of the substrates tested. The K(m) values of sucrose/raffinose/stachyose were 1.7/1.8/5.0 mM for PaxgINV1 and 1.6/1.7/1.9 mM for PaxgINV2, respectively. Activity analyses in the presence of various metal cations showed that PaxgINV2 was strongly inhibited by Cu(2+), Zn(2+) and Hg(2+), while PaxgINV1 was only weakly inhibited by these cations. The results from this study, coupled with previous expression data, suggest that PaxgINV1 and PaxgINV2 have distinct roles with respect to the physiology and development of hybrid poplar, specifically phloem unloading and processes related to dormancy and bud break.
先前研究表明，两种杂种杨树细胞壁转化酶（EC 3.2.1.26；PaxgINV1和PaxgINV2）在营养组织中受到时空调控。PaxgINV1的表达与休眠相关过程有关，而PaxgINV2在生长和扩张的组织中表达显著。为了进一步阐明这些关键细胞壁酶的生理作用，PaxgINV1和PaxgINV2在甲基营养酵母毕赤酵母中进行了异源表达。杨树转化酶的三维预测模型显示，一个结构通道同时包含保守的β-呋喃果糖苷酶和细胞壁转化酶基序，表明该通道是这些酶的假定活性位点。重组PaxgINV1和PaxgINV2的最适pH分别为4.8和5.6，最适温度分别为45℃和40℃。功能表征显示，这两种酶都能够水解蔗糖、棉子糖、水苏糖和毛蕊花糖的果糖残基，PaxgINV2对每种测试底物具有更高的比活性。PaxgINV1的蔗糖/棉子糖/水苏糖的K(m)值分别为1.7/1.8/5.0 mM，PaxgINV2的分别为1.6/1.7/1.9 mM。在各种金属阳离子存在下的活性分析表明，PaxgINV2受到Cu(2+)、Zn(2+)和Hg(2+)的强烈抑制，而PaxgINV1仅受到这些阳离子的微弱抑制。本研究结果与先前的表达数据表明，PaxgINV1和PaxgINV2在杂种杨树的生理和发育方面具有不同作用，特别是在韧皮部卸载以及与休眠和芽萌发相关的过程中。
