Abstract
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.
Full Text
The Full Text of this article is available as a PDF (1.0 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Blanchard J. S., Englard S., Kondo A. gamma-Butyrobetaine hydroxylase: a unique protective effect of catalase. Arch Biochem Biophys. 1982 Dec;219(2):327–334. doi: 10.1016/0003-9861(82)90163-1. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
- Britsch L. Purification and characterization of flavone synthase I, a 2-oxoglutarate-dependent desaturase. Arch Biochem Biophys. 1990 Oct;282(1):152–160. doi: 10.1016/0003-9861(90)90099-k. [DOI] [PubMed] [Google Scholar]
- Cunliffe C. J., Franklin T. J., Hales N. J., Hill G. B. Novel inhibitors of prolyl 4-hydroxylase. 3. Inhibition by the substrate analogue N-oxaloglycine and its derivatives. J Med Chem. 1992 Jul 10;35(14):2652–2658. doi: 10.1021/jm00092a016. [DOI] [PubMed] [Google Scholar]
- De Jong L., Kemp A. Stoicheiometry and kinetics of the prolyl 4-hydroxylase partial reaction. Biochim Biophys Acta. 1984 May 31;787(1):105–111. doi: 10.1016/0167-4838(84)90113-4. [DOI] [PubMed] [Google Scholar]
- Hashimoto T., Yamada Y. Purification and characterization of hyoscyamine 6 beta-hydroxylase from root cultures of Hyoscyamus niger L. Hydroxylase and epoxidase activities in the enzyme preparation. Eur J Biochem. 1987 Apr 15;164(2):277–285. doi: 10.1111/j.1432-1033.1987.tb11055.x. [DOI] [PubMed] [Google Scholar]
- Katz E., Kamal F., Mason K. Biosynthesis of trans-4-hydroxy-L-proline by Streptomyces griseoviridus. J Biol Chem. 1979 Jul 25;254(14):6684–6690. [PubMed] [Google Scholar]
- Kaule G., Günzler V. Assay for 2-oxoglutarate decarboxylating enzymes based on the determination of [1-14C]succinate: application to prolyl 4-hydroxylase. Anal Biochem. 1990 Feb 1;184(2):291–297. doi: 10.1016/0003-2697(90)90683-z. [DOI] [PubMed] [Google Scholar]
- Kondo A., Blanchard J. S., Englard S. Purification and properties of calf liver gamma-butyrobetaine hydroxylase. Arch Biochem Biophys. 1981 Dec;212(2):338–346. doi: 10.1016/0003-9861(81)90374-x. [DOI] [PubMed] [Google Scholar]
- Kuttan R., Radhakrishnan A. N. Biochemistry of the hydroxyprolines. Adv Enzymol Relat Areas Mol Biol. 1973;37:273–347. doi: 10.1002/9780470122822.ch5. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Majamaa K., Hanauske-Abel H. M., Günzler V., Kivirikko K. I. The 2-oxoglutarate binding site of prolyl 4-hydroxylase. Identification of distinct subsites and evidence for 2-oxoglutarate decarboxylation in a ligand reaction at the enzyme-bound ferrous ion. Eur J Biochem. 1984 Jan 16;138(2):239–245. doi: 10.1111/j.1432-1033.1984.tb07907.x. [DOI] [PubMed] [Google Scholar]
- Miles E. W. Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol. 1977;47:431–442. doi: 10.1016/0076-6879(77)47043-5. [DOI] [PubMed] [Google Scholar]
- Myllylä R., Günzler V., Kivirikko K. I., Kaska D. D. Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases. Biochem J. 1992 Sep 15;286(Pt 3):923–927. doi: 10.1042/bj2860923. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Myllylä R., Kuutti-Savolainen E. R., Kivirikko K. I. The role of ascorbate in the prolyl hydroxylase reaction. Biochem Biophys Res Commun. 1978 Jul 28;83(2):441–448. doi: 10.1016/0006-291x(78)91010-0. [DOI] [PubMed] [Google Scholar]
- Myllylä R., Majamaa K., Günzler V., Hanauske-Abel H. M., Kivirikko K. I. Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase. J Biol Chem. 1984 May 10;259(9):5403–5405. [PubMed] [Google Scholar]
- Ng S. F., Hanauske-Abel H. M., Englard S. Cosubstrate binding site of Pseudomonas sp. AK1 gamma-butyrobetaine hydroxylase. Interactions with structural analogs of alpha-ketoglutarate. J Biol Chem. 1991 Jan 25;266(3):1526–1533. [PubMed] [Google Scholar]
- Onishi M., Okumura Y., Okamoto R., Ishikura T. Proline hydroxylation by cell free extract of a streptomycete. Biochem Biophys Res Commun. 1984 Apr 16;120(1):45–51. doi: 10.1016/0006-291x(84)91411-6. [DOI] [PubMed] [Google Scholar]
- Puistola U., Turpeenniemi-Hujanen T. M., Myllylä R., Kivirikko K. I. Studies on the lysyl hydroxylase reaction. I. Initial velocity kinetics and related aspects. Biochim Biophys Acta. 1980 Jan 11;611(1):40–50. doi: 10.1016/0005-2744(80)90040-6. [DOI] [PubMed] [Google Scholar]
- Roach P. L., Clifton I. J., Fülöp V., Harlos K., Barton G. J., Hajdu J., Andersson I., Schofield C. J., Baldwin J. E. Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes. Nature. 1995 Jun 22;375(6533):700–704. doi: 10.1038/375700a0. [DOI] [PubMed] [Google Scholar]
- Wichmann C. F., Liesch J. M., Schwartz R. E. L-671,329, a new antifungal agent. II. Structure determination. J Antibiot (Tokyo) 1989 Feb;42(2):168–173. doi: 10.7164/antibiotics.42.168. [DOI] [PubMed] [Google Scholar]
- Zhang Z., Schofield C. J., Baldwin J. E., Thomas P., John P. Expression, purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from tomato in Escherichia coli. Biochem J. 1995 Apr 1;307(Pt 1):77–85. doi: 10.1042/bj3070077. [DOI] [PMC free article] [PubMed] [Google Scholar]