Biochemical and molecular biological characterisation of chalcone 3-hydroxylase

Abstract

The recently discovered cytochrome P450 depended enzyme CH3H catalyses the hydroxylation in position 3 of the B ring of chalcones. This reaction is similar to the hydroxylation of flavonoids at position 3' but cannot be catalysed by the well-known F3-H despite the broad substrate specificity of this enzyme. The objective of this work was the construction of vectors carrying F3-H and CH3H cDNA clones for the production of Strep-tagged fusion proteins, comparison of the sequences to identify putative CH3H specific sites, Mutation of the cDNA clones and testing the substrate specificity of the resulting fusion proteins and identification of specific regions in the amino acid sequence of CH3H determining the substrate specificity. Strep-tagged fusion proteins were produced from CH3H of Cosmos sulphureus, Bidens ferulifolia and Coreopsis grandiflora and F3-H of Bidens ferulifolia and Dahlia variabilis for a better purification of the recombinant enzymes. Mutations were performed in CH3Hs and F3-H of Bidens ferulifolia, Cosmos sulphureus, Dahlia variabilis and putative CH3H of Coreopsis grandiflora. The mutations at position 104 in SRS1 and 219 in SRS2 in Cosmos CH3H had characteristic influence on substrate specificity. Changes in these positions converted CH3H into F3-H. The selected amino acids for mutation in Bidens CH3H and F3-H did not have any influence on substrate specificity and all recombinant enzymes were still CH3H-s. After mutation of the inactive, putative CH3H of Coreopsis catalytic activity was observed for the first time. The mutants 53Q and 104A in SRS 1 and 211M in SRS 2 seem to be authentic CH3Hs.