Insights into the catalytic properties of bamboo vacuolar invertase through mutational analysis of active site residues.
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.
Chen
Tai-Hung
TH
Institute of Microbiology and Biochemistry and Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan.
Huang
Yu-Chiao
YC
Yang
Chii-Shen
CS
Yang
Chien-Chih
CC
Wang
Ai-Yu
AY
Sung
Hsien-Yi
HY
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018506:Gene Expression Regulation, Plant; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000378:metabolism; D006109:Poaceae / Q000201:enzymology*; D011487:Protein Conformation; D043324:beta-Fructofuranosidase / Q000235:genetics* / Q000378:metabolism*
D010940:Plant Proteins; D043324:beta-Fructofuranosidase