An isozyme of the NADP-malic enzyme of a CAM plant, Aloe arborescens, with variation on conservative amino acid residues

In Aloe arborescens, an obligate CAM plant, Western analysis detected three major isoforms of NADP-malic enzyme (NADP-ME), 72 kDa with a pI of 6.0, 65 kDa with a pi of 5.6 and 65 kDa with a pI of 5.5. Among them, the 65 kDa protein with a pI of 5.5 was leaf-specific, and the 65 kDa protein with a pI of 5.6 was found only in roots, whereas the 72 kDa protein was uniformly detected in both organs. Activity staining indicated enzyme activity of both 65 kDa NADP-MEs but little activity of the 72 kDa protein. A cDNA clone encoding a leaf-abundant NADP-ME, AME1, was isolated. Deduced amino acid sequence of AME1 showed a high degree of homology to known NADP-MEs, but it was also found that AME1 contained substitutions on five conservative amino acid residues, some of which have been predicted to be important for their enzyme activity. Transgenic rice carrying the aloe AME1 gene efficiently produced an additional 65 kDa protein with a pI of 5.5 as an active NADP-ME. These results indicate that AME1 corresponds to the leaf-specific 65 kDa NADP-ME, which may be involved in CAM photosynthesis. It was also shown that substitutions of these conservative amino acid residues identified in AME1 still allowed it to give enzyme activity. (C) 2000 Elsevier Science B.V. All rights reserved.