The recognition of glycolate oxidase apoprotein with flavin analogs in higher plants.
The dependence of glycolate oxidase apoprotein (apoGO) activity on flavin analogs was surveyed in 9 higher plants from 7 families. Activities of all apoGOs depended not only on flavin mononucleotide (FMN) but also on flavin adenine dinucleotide (FAD), but not on riboflavin. The kinetic analysis showed that FMN was the optimum cofactor for apoGO from leaves of Brassica campestris. In plant kingdom, FMN, FAD and riboflavin are three flavin analogs with very similar structure, and they could coexist and be inter-converted from each other, so the question is how the apoprotein of glycolate oxidase (GO) recognized these flavin analogs. No inhibition effect of riboflavin on the activity of apoGO with FMN or FAD was found and no obvious quenching of riboflavin or apoGO protein fluorescence was detected with the addition of apoGO or riboflavin, respectively. These results indicated that riboflavin did not bind to apoGO tightly like FMN and FAD. Inorganic phosphate (Pi) did inhibit the activity of GO, and kinetic analysis revealed that this inhibition was caused by the competitive binding to apoGO between Pi and FMN. This competitive binding was further confirmed by the inhibition of Pi to the quenching of FMN and apoGO protein fluorescence with apoGO and FMN, respectively. It was suggested that the 5'-phosphate group of FMN or FAD may play a key role in the recognition and binding of riboflavin analog cofactors with apoGO.
Wang
Wei-Jun
WJ
College of Life Science, South China Agricultural University, Guangzhou. wangweijun@scau.edu.cn
Huang
Jing-Quan
JQ
Yang
Chong
C
Huang
Jiu-Jiu
JJ
Li
Ming-Qi
MQ
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001667:Binding, Competitive; D001937:Brassica / Q000201:enzymology; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D005415:Flavins / Q000737:chemistry*; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D018515:Plant Leaves / Q000737:chemistry; D010944:Plants / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D001059:Apoproteins; D005415:Flavins; D010710:Phosphates; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase