Abstract
The enzyme, desacetoxyvindoline 4-hydroxylase, was purified to apparent homogeneity from Catharanthus roseus by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, green 19-agarose, hydroxylapatite, α-kg sepharose and Mono Q. The 4-hydroxylase was characterized by its strict specificity for position 4 of desacetoxyvindoline suggesting it to catalyze the second to last step in vindoline biosynthesis. The molecular mass of the native and denatured 4-hydroxylase was 45 kDa and 44.7 kDa, respectively, suggesting that the native enzyme is a monomer. Two-dimensional isoelectric focusing under denaturing conditions resolved the purified 4-hydroxylase into three charge isoforms of pIs 4.6, 4.7 and 4.8. The purified 4-hydroxylase exhibited no requirement for divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. The enzyme was not inhibited by EDTA or SH-group reagents at concentrations up to 10 mM. The mechanism of action of desacetoxyvindoline 4-hydroxylase was investigated. The results of substrate interaction kinetics and product inhibition studies suggest an Ordered Ter Ter mechanism where α-kg is the first substrate to bind followed by the binding of O2 and desacetoxyvindoline. Their K m values for α-kg, O2 and desacetoxyvindoline are 45 μM, 45 μM and 0.03 μM, respectively. The first product to be released was deacetylvindoline followed by CO2 and succinate, respectively.
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Abbreviations
- α-kg:
-
α-ketoglutarate or 2-oxoglutarate
- NMT:
-
N-methyltransferase
- SAM:
-
S-adenosyl-l-methionine
- TLC:
-
thin layer chromatography
- VBL:
-
vinblastine
- VCR:
-
vincristine
References
Balsevich J, De Luca V & Kurz WGW (1986) Altered alkaloid pattern in dark grown seedlings of Catharanthus roseus. The isolation and characterization of 4-desacetoxyvindoline: a novel indole alkaloid and proposed precursor of vindoline. Heterocycles 24: 2415–2421
Cleland WW (1967) Steady state kinetics. In: Boyer PD (Ed) The Enzymes. Vol II (pp 1–61) Academic Press, New York
Cleveland DW, Fisher SG, Kirscher MW & Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis of gel electrophoresis. J. Biol. Chem. 252: 1102–1106
De Carolis E, Chan F, Balsevich J & De Luca V (1990) Isolation and characterization of a 2-oxoglutarate-dependent dioxygenase involved in the second last step in vindoline biosynthesis. Plant Physiol. 94: 1323–1329
De Carolis E & De Luca V (1993) Purification, characterization and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from Catharanthus roseus. J. Biol. Chem. 268: 5504–5511
De Luca V (1993) Enzymology of indole alkaloid biosynthesis. In: Lea PJ (Ed) Methods in Plant Biochemistry, Vol 9 (pp 345–368), Academic Press, New York
De Luca V & Kurz WGW (1988) Monoterpene indole alkaloids (Catharanthus alkaloids). In: Constable F & Vasil IK (Eds) Cell Culture and Somatic Genetics of Plants, Vol 5 (pp 385–401). Academic Press, San Diego
Dethier M & De Luca V (1993) Partial purification of an N-methyltransferase involved in vindoline biosynthesis in Catharanthus roseus. Phytochemistry 32: 673–678
Holme E (1975) A kinetic study of thymine 7-hydroxylase from Neurospora crassa Biochemistry 14: 4999–5003
Myllyla R, Tuderman L & Kivirikko KI (1977) Mechanism of prolyl hydroxylase reaction: kinetic analysis of the reaction sequence. Eur. J. Biochem. 80: 349–357
Power R, Kurz WGW & De Luca V (1990) Purification and characterization of acetylcoenzyme A: deacetyvindoline 4- acetyltransferase from Catharanthus roseus. Arch. Biochem. Biophys. 279: 370–376
Puistola U, Turpeenniemi-Hujanen TM, Myllyla R & Kivirikko KI (1980) Studies on the lysyl hydroxylase reaction: Inhibition kinetics and the reaction mechanism. Biochim. Biophys. Acta 611: 51–60
Rhoads RE & Udenfriend S (1968) Decarboxylation of α-ketoglutarate coupled to collagen proline hydroxylase. Proc. Natl. Acad. Sci. 60: 1473–1478
Robinson T (1981) General theories of alkaloid biosynthesis. In: Kleinzeller A, Springer GF & Wittmann HG (Eds) The biochemistry of alkaloids: Molecular biology biochemistry and biophysics. Vol II (pp 12–136). Springer-Verlag, Berlin
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De Carolis, E., De Luca, V. A novel 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis: characterization, purification and kinetic properties. Plant Cell Tiss Organ Cult 38, 281–287 (1994). https://doi.org/10.1007/BF00033888
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DOI: https://doi.org/10.1007/BF00033888