PURIFICATION AND INITIAL CHARACTERIZATION OF PROLINE 4-HYDROXYLASE FROM STREPTOMYCES GRISEOVIRIDUS-P8648 - A 2-OXOACID, FERROUS-DEPENDENT DIOXYGENASE INVOLVED IN ETAMYCIN BIOSYNTHESIS

Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenas e involved in the biosynthesis of the secondary metabolite etamycin. T he purification, in low yield, of proline 4-hydroxylase from Streptomy ces griseoviridus P8648 to near apparent homogeneity and its initial c haracterization are reported. In most respects proline 4-hydroxylase i s a typical member of the 2-oxoacid-dependent dioxygenase family. It i s monomeric (M(r) approx. 38000) (by gel filtration on Superdex-G75) a nd has typically strict requirements for ferrous ion and 2-oxoglutarat e. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. L -Proline-uncoupled turnover of 2-oxoglutarate to succinate and CO2 was observed. The addition of L-ascorbate did not stimulate L-proline-cou pled turnover of 2-oxoglutarate, but did stimulate L-proline-uncoupled turnover. L-Ascorbate caused a time-dependent inhibition of L-proline hydroxylation, The enzyme was completely inactivated by preincubation with diethyl pyrocarbonate under histidine-modifying conditions. This inactivation could be partially prevented by the inclusion of L-proli ne and 2-oxoglutarate in the preincubation mixture, suggesting the pre sence of histidine residue(s) at the active site.