A new prolyl hydroxylase acting on poly-L-proline, from suspension cultured cells of Vinca rosea.

A new prolyl hydroxylase having a novel substrate specificity was isolated from the suspension-cultured cells of Vinca rosea. This enzyme was solubilized with 0.05 M Tris-HCl buffer (pH 7.4) containing 0.1% Triton X-100, 0.3 M NaCl and 0.5 mM beta-mercaptoethanol from the membrane fractions of the cells, and was partially purified by (NH4)2SO4 fractionation and DEAE-Sephadex A-50 column chromatography. The enzyme preparation was found to require O2, Fe2+, ascorbate, alpha-ketoglutarate and poly-L-proline to attain maximum activity. The plant enzyme does not hydroxylate free proline and di-, tri- and tetra-L-proline, but hydroxylates octa-L-proline and poly-L-proline (Mr greater than 2000). Model peptides of unhydroxylated collagen, (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 are poor substrates for the plant enzyme. This means that the plant enzyme has a novel substrate specificity in regard to peptidyl substrate, and this differs from vertebrate prolyl hydroxylase, proline,2-oxoglutarate dioxygenase (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase, EC 1.14.11.2).