Elucidation of active site residues of Arabidopsis thaliana flavonol synthase provides a molecular platform for engineering flavonols.
Arabidopsis thaliana flavonol synthase (aFLS) catalyzes the production of quercetin, which is known to possess multiple medicinal properties. aFLS is classified as a 2-oxoglutarate dependent dioxygenase as it requires ferrous iron and 2-oxoglutarate for catalysis. In this study, the putative residues for binding ferrous iron (H221, D223 and H277), 2-oxoglutarate (R287 and S289) and dihydroquercetin (H132, F134, K202, F293 and E295) were identified via computational analyses. To verify the proposed roles of the identified residues, 15 aFLS mutants were constructed and their activities were examined via a spectroscopic assay designed in this study. Mutations at H221, D223, H277 and R287 completely abolished enzymes activities, supporting their importance in binding ferrous iron and 2-oxoglutarate. However, mutations at the proposed substrate binding residues affected the enzyme catalysis differently such that the activities of K202 and F293 mutants drastically decreased to approximately 10% of the wild-type whereas the H132F mutant exhibited approximately 20% higher activity than the wild-type. Kinetic analyses established an improved substrate binding affinity in H132F mutant (Km: 0.027+/-0.0028 mM) compared to wild-type (Km: 0.059+/-0.0063 mM). These observations support the notion that aFLS can be selectively mutated to improve the catalytic activity of the enzyme for quercetin production.
Chua
Chun Song
CS
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore.
Biermann
Daniela
D
Goo
Kian Sim
KS
Sim
Tiow-Suan
TS
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001665:Binding Sites; D001709:Biotechnology; D002384:Catalysis; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011794:Quercetin / Q000096:biosynthesis* / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016415:Sequence Alignment
D003067:Coenzymes; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D011794:Quercetin; D010088:Oxidoreductases; C085313:flavonol synthase