Vacuolar invertases (VINs) from Cu-tolerant and non-tolerant populations of Elsholtzia haichowensis have similar enzyme properties, and the enzyme protein divergences contribute little to the varied VIN activities between the contrasting populations. In our previous studies of Elsholtzia haichowensis, vacuolar invertase (VIN) activity in roots of a Cu-tolerant population was found to be significantly higher than that of a non-tolerant population under Cu stress. Divergences of amino acid residues in a sucrose-binding box and other regions of the VINs were detected. To test whether the amino acid divergences influence the enzyme properties of VINs, and thus are relevant to the differences in enzyme activities between the contrasting populations of E. haichowensis, two VIN genes from the Cu-tolerant population (EhCvINV) and non-tolerant population (EhNvINV) were heterologously expressed in Pichia pastoris, and the enzyme properties of the recombinants were characterized and compared. Both of the recombinant enzymes showed temperature optima of 70 °C and pH optima of 4.5-5.5. Copper as well as other heavy metals caused almost the same inhibition to EhNvINV and EhCvINV. No statistically significant differences were observed between EhNvINV and EhCvINV in K m and k cat values for sucrose. The results provided evidence that the observed residue divergences had little influence on the enzyme properties of VIN in E. haichowensis, and the varied VIN activities between the contrasting populations under Cu stress were not relevant to the amino acid divergences in the proteins. Also, some other possible reasons accounting for this difference in invertase activities were discussed, such as up-regulation of expression of the EhCvINV gene under Cu stress, as Cu tolerance mechanisms in Cu-mine plants.
Keywords: Amino acid divergences; Cu; Elsholtzia haichowensis; Heterologous expression; Vacuolar invertases.