A single amino acid in the PSPG-box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus.
Curcumin glucosyltransferase (CaUGT2) isolated from cell cultures of Catharanthus roseus exhibits unique substrate specificity. To identify amino acids involved in substrate recognition and catalytic activity of CaUGT2, a combination of domain swapping and site-directed mutagenesis was carried out. Exchange of the PSPG-box of CaUGT2 with that of NtGT1b (a phenolic glucosyltransferase from tobacco) led to complete loss of enzyme activity in the resulting recombinant protein. However, replacement of Arg378 of the NtGT1b PSPG-box with cysteine, the corresponding amino acid in CaUGT2, restored the catalytic activity of the chimeric enzyme. Further site-directed mutagenesis revealed that the size of the amino acid side-chain in that particular site is critical to the catalytic activity of CaUGT2.
Masada
Sayaka
S
Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori 3-1, Nagoya, Japan.
Terasaka
Kazuyoshi
K
Mizukami
Hajime
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D030008:Catharanthus / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D003474:Curcumin / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D009596:Nitrophenols; D010940:Plant Proteins; D001120:Arginine; D005964:Glucosyltransferases; D003474:Curcumin; D003545:Cysteine; C024836:4-nitrophenol