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 (Mr approx. 38000) (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.
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January 1996
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Research Article|
January 01 1996
Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis Available to Purchase
Christopher C. LAWRENCE;
Christopher C. LAWRENCE
1The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, South Parks Road, Oxford OX1 3QY, U.K.
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Wendy J. SOBEY;
Wendy J. SOBEY
1The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, South Parks Road, Oxford OX1 3QY, U.K.
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Jack E. BALDWIN;
Jack E. BALDWIN
1The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, South Parks Road, Oxford OX1 3QY, U.K.
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Christopher J. SCHOFIELD
Christopher J. SCHOFIELD
1The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, South Parks Road, Oxford OX1 3QY, U.K.
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Publisher: Portland Press Ltd
Received:
August 11 1995
Accepted:
August 25 1995
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 313 (1): 185–191.
Article history
Received:
August 11 1995
Accepted:
August 25 1995
Citation
Christopher C. LAWRENCE, Wendy J. SOBEY, Robert A. FIELD, Jack E. BALDWIN, Christopher J. SCHOFIELD; Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis. Biochem J 1 January 1996; 313 (1): 185–191. doi: https://doi.org/10.1042/bj3130185
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