Insights into the catalytic properties of bamboo vacuolar invertase through mutational analysis of active site residues
Resource
Phytochemistry 70 (1): 25-31
Journal
Phytochemistry
Journal Volume
70
Journal Issue
1
Pages
25-31
Date Issued
2009
Date
2009
Author(s)
Abstract
Plant acid invertases, which are either associated with the cell wall or present in vacuoles, belong to family 32 of glycoside hydrolases (GH32). Homology modeling of bamboo vacuolar invertase Bobetafruct3 using Arabidopsis cell-wall invertase AtcwINV1 as a template showed that its overall structure is similar to GH32 enzymes, and that the three highly conserved motifs, NDPNG, RDP and EC, are located in the active site. This study also used site-directed mutagenesis to examine the roles of the conserved amino acid residues in these three motifs, which include Asp135, Arg259, Asp260, Glu316 and Cys317, and a conserved Trp residue (Trp159) that resides between the NDPNG and RDP motifs. The mutants W159F, W159L, E316Q and C317A retained acid invertase activity, but no invertase activity was observed for the mutant E316A or mutants with changes at Asp135, Arg259, or Asp260. The apparent K(m) values of the four mutants with invertase activity were all higher than that of the wild-type enzyme. The mutants W159L and E316Q exhibited lower k(cat) values than the wild-type enzyme, but an increase in the k(cat) value was observed for the mutants W159F and C317A. The results of this study demonstrate that these residues have individual functions in catalyzing sucrose hydrolysis.
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journal article
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