Single amino acid mutations of Medicago glycosyltransferase UGT85H2 enhance activity and impart reversibility.
The glycosyltransferase UGT85H2 from Medicago truncatula catalyzes glucosylation of the (iso)flavonoids kaempferol and biochanin A. Structure-based mutagenesis of UGT85H2 was carried out to explore the roles of amino acids involved in substrate binding. Substitution of Ile305 by threonine increased catalytic efficiency 37- or 19-fold with kaempferol or biochanin A as acceptor, respectively. A point mutation V200E also dramatically improved the turnover rate and catalytic efficiency by 15-fold for kaempferol and 54-fold for biochanin A. More interestingly, this single mutation (V200E) conferred reversibility in the glycosyltransfer reaction, indicating that Glu200 is a key determinant for the deglycosylation function.
Modolo
Luzia V
LV
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA.
Escamilla-Treviño
Luis L
LL
Dixon
Richard A
RA
Wang
Xiaoqiang
X
D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics; D019833:Genistein / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D044949:Kaempferols / Q000378:metabolism; D007700:Kinetics; D046913:Medicago truncatula / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D044949:Kaempferols; D011994:Recombinant Proteins; C006552:kaempferol; D019833:Genistein; D016695:Glycosyltransferases; C004541:biochanin A