Mutation analysis of violaxanthin de-epoxidase identifies substrate-binding sites and residues involved in catalysis.
Plants are able to deal with variable environmental conditions; when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carotenoid violaxanthin is converted to zeaxanthin by violaxanthin de-epoxidase, using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the violaxanthin de-epoxidase catalytic domain, we identified the putative binding sites for violaxanthin and ascorbate by in silico docking. Amino acid residues lying in close contact with the two substrates were analyzed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate-binding sites and point to two residues, Asp-177 and Tyr-198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed.
Dipartimento di Biologia, Università di Padova, Via Ugo Bassi 58 B, 35121 Padova, Italy.
Giorgia
G
Saga
Alejandro
A
Giorgetti
Christian
C
Fufezan
Giorgio M
GM
Giacometti
Roberto
R
Bassi
Tomas
T
Morosinotto
D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004252:DNA Mutational Analysis; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry*; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry; D024341:Xanthophylls / Q000737:chemistry; D065146:Zeaxanthins
D024341:Xanthophylls; D065146:Zeaxanthins; D001224:Aspartic Acid; D014443:Tyrosine; D010088:Oxidoreductases; C023290:violaxanthin de-epoxidase; D001205:Ascorbic Acid