Functional assignment of Glu386 and Arg388 in the active site of L-galactono-gamma-lactone dehydrogenase.
The flavoenzyme L-galactono-gamma-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plants. Little is known about the catalytic mechanism of GALDH and related aldonolactone oxidoreductases. Here we identified an essential Glu-Arg pair in the active site of GALDH from Arabidopsis thaliana. Glu386 and Arg388 variants show high K(m) values for L-galactono-1,4-lactone and low turnover rates. Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor. Glu386 is involved in productive substrate binding. The E386D variant has lost its specificity for L-galactono-1,4-lactone and shows the highest catalytic efficiency with L-gulono-1,4-lactone.
Leferink
Nicole G H
NG
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. m.f.jose@rug.nl
Jose
Mac Donald F
MD
van den Berg
Willy A M
WA
van Berkel
Willem J H
WJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D007783:Lactones / Q000378:metabolism*; D008969:Molecular Sequence Data; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism*; D013400:Sugar Acids / Q000378:metabolism*
D007783:Lactones; D013400:Sugar Acids; C534014:galactono-1,4-lactone; D018698:Glutamic Acid; D001120:Arginine; D044925:Oxidoreductases Acting on CH-CH Group Donors; C093910:galactonolactone dehydrogenase