Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis.

Journal article

Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseoviridus P8648 to near, apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (M(r) approx. 38,000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. 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-coupled 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-proline and 2-oxoglutarate in the preincubation mixture, suggesting the presence of histidine residue(s) at the active site.

Authors: Lawrence, C; Sobey, W; Field, R; Baldwin, J; Schofield, C

• Publication status: Published
• Journal: Biochemical journal
• Volume: 313 ( Pt 1)
• Issue: 1
• Pages: 185-191
• Publication date: 1996-01-01
• EISSN: 1470-8728
• ISSN: 0264-6021
• Language: English
• Keywords: Ketoglutaric Acids; Enzyme Inhibitors; Ascorbic Acid; Anti-Bacterial Agents; Substrate Specificity; Procollagen-Proline Dioxygenase; Streptomyces; Diethyl Pyrocarbonate; Ions; Macrolides; Peptides; Ferrous Compounds