Heterologous expression and functional characterization of two hybrid poplar cell-wall invertases

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 β-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°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 Cu²⁺, Zn²⁺ and Hg²⁺, 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.