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 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.
Lawrence
C C
CC
Dyson Perrins Laboratory, Oxford, UK.
Sobey
W J
WJ
Field
R A
RA
Baldwin
J E
JE
Schofield
C J
CJ
D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001205:Ascorbic Acid / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005296:Ferrous Compounds / Q000378:metabolism*; D007477:Ions; D007656:Ketoglutaric Acids / Q000378:metabolism*; D018942:Macrolides / Q000378:metabolism; D010455:Peptides; D011393:Procollagen-Proline Dioxygenase / Q000302:isolation & purification* / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D047630:Depsipeptides
D000900:Anti-Bacterial Agents; D001205:Ascorbic Acid; D004047:Diethyl Pyrocarbonate; D004791:Enzyme Inhibitors; D005296:Ferrous Compounds; D007477:Ions; D007656:Ketoglutaric Acids; D018942:Macrolides; D010455:Peptides; D011393:Procollagen-Proline Dioxygenase; C004910:etamycin; D047630:Depsipeptides