Abstract
Uridine 5′-diphosphoglucose-dependent glucosyl-transferases (UDP-glucose:betanidin 5-O- and 6-O-glucosyltransferases; 5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific transfer of glucose to the 5- and 6-hydroxy group of betanidin in the formation of betanin and gomphrenin I, respectively. Both GT activities were partially purified from cell suspension cultures of Dorotheanthus bellidiformis (Burm. f.) N.E. Br. Isoelectric focusing of crude protein extracts indicated the presence of three 5-GT isoforms and a single 6-GT form. The 5-GT isoforms were partially separated from each other and completely from the 6-GT. Studies of the glucosyltransferase activities were focused on the major isoform of the 5-GTs and the 6-GT, which displayed the same pH optimum near 7.5 in K-phosphate buffer. A 3- and 2.5-fold enrichment and 11% and 10% recovery of the 5-GT and 6-GT, respectively, were routinely achieved; however, a 3300-fold enrichment of the major 5-GT isoform and a 6-fold enrichment of the 6-GT were also achieved. Both enzymes are monomers and displayed apparent native Mrs near 55 000. The maxima of the reaction temperature were at 50 °C for the 5-GT and at 37°C for the 6-GT with respective apparent energies of activation of 51 and 53 kJ · mol−1. Kinetic studies indicated that the apparent Michaelis constants (apparent K m) of the GTs for one substrate were dependent on the concentration of the second substrate. However, the relationship between the apparent K m values and the dissociation constants (K i) were different; m > K i applies for the 5-GT and K m < K i for the 6-GT activity. Consequently, this results in a predominant formation of betanin at low substrate concentrations, but a predominant formation of gomphrenin I at high substrate concentrations, assuming that both enzymes may compete freely for their substrates. This might explain why we could not observe a correlation between extractable 5-GT and 6-GT activities and the in-vivo accumulation of the respective products from cell-suspension cultures of D. bellidiformis.
Similar content being viewed by others
Abbreviations
- BSA:
-
bovine serum albumin
- DOPA:
-
l-(3,4-dihydroxyphenyl)-alanine
- 5-GT:
-
UDP-glucose:betanidin 5-O-glucosyltransferase
- 6-GT:
-
UDP-glucose:betanidin 6-O-glucosyl-transferase
- Mr :
-
molecular mass
References
Bisswanger H (1994) Enzymkinetik. Theorie und Methoden (2. Aufl.). VCH, Weinheim
Böhm H, Böhm L, Rink E (1991) Establishment and characterization of a betaxanthin-producing cell culture from Portulaca grandiflora. Plant Cell Tissue Organ Cult 26: 75–82
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–354
Girod P-A, Zryd J-P (1991) Biogenesis of betalains: purification and partial characterization of DOPA-4,5-dioxygenase from Amanita muscaria. Phytochemistry 30: 169–174
Heller W, Forkmann G (1988) Biosynthesis. In: Harborne J/B (ed) The flavonoids. Advances in research since 1980. Chapman and Hall, London, pp 399–425
Heller W, Forkmann G (1994) Biosynthesis. In: Harborne JB (ed) The flavonoids. Advances in research since 1986. Chapman and Hall, London, pp 499–535
Heuer S, Strack D (1992) Synthesis of betanin from betanidin and UDP-glucose by a protein preparation from cell suspension cultures of Dorotheanthus bellidiformis (Burm. f) N.E. Br. Planta 186: 626–628
Heuer S, Wray V, Metzger JW, Strack D (1992) Betacyanins from flowers of Gomphrena globosa. Phytochemistry 31: 1801–1807
Heuer S, Richter S, Metzger JW, Wray V, Nimtz M, Strack D (1994) Betacyanins from bracts of Bougainvillea glabra. Phytochemistry 37: 761–767
Kho KFF, Kamsteeg J, van Brederode J (1978) Identification, properties and genetic control of UDP-glucose:cyanidin 3-O- glucosyltransferase in Petunia hybrida. Z Pflanzenphysiol 88: 449–464
Lämmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100–127
Mock H-P, Strack D (1993) Energetics of the uridine 5′-diphospho-glucose: hydroxycinnamic acid acyl-glucosyltransferase reaction. Phytochemistry 32: 575–579
Robertson EF, Dannelly HK, Malloy PJ, Reeves HC (1987) Rapid isoelectric focusing in a vertical polyacrylamide minigel system. Anal Biochem 167: 290–294
Segel IH (1975) Enzyme kinetics. Behaviour and analysis of rapid equilibrium and steady-state enzyme systems. John Wiley & Sons, New York, Chichester, Brisbane, Toronto
Steglich W, Strack D (1990) Betalains. In: Brossi A (ed) The alkaloids. Chemistry and pharmacology, vol 39, Academic Press, London, pp 1–62
Strack D, Wray V (1989) Anthocyanins. Methods Plant Biochem 1: 325–356
Strack D, Steglich W, Wray V (1993) Betalains. Methods Plant Biochem 8: Alkaloids and sulphur Compounds.421–450
Terradas F, Wyler H (1991a) 2,3- and 4,5- Secodopas, the biosynthetic intermediates generated from l-dopa by an enzyme system extracted from the fly agaric, Amanita muscaria L., and their spontaneous conversion to muscaflavin and betalamic acid, respectively, and betalains. Helv Chim Acta 74: 124–140
Terradas F, Wyler H (1991b) The secodopas, natural pigments in Hygrocybe conica and in Amanita muscaria. Phytochemistry 30: 3251–3253
Wilcox ME, Wyler H, Dreiding AS (1965) Stereochemie von Betanidin und Isobetanidin. Helv Chim Acta 48: 1134–1147
Wyler H, Dreiding AS (1957) Kristallisiertes Betanin. Helv Chim Acta 40: 191–192
Wyler H, Dreiding AS (1959) Darstellung und Abbauprodukte des Betanidins. Helv Chim Acta 42: 1699–1702
Wyler H, Vincenti G, Mercier M, Sassu G, Dreiding AS (1959) Zur Konstitution des Rindenfarbstoffes Betanin. Helv Chim Acta 42: 1696–1698
Author information
Authors and Affiliations
Additional information
This investigation was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
Rights and permissions
About this article
Cite this article
Heuer, S., Vogt, T., Böhm, H. et al. Partial purification and characterization of UDP-glucose: betanidin 5-O- and 6-O-glucosyltransferases from cell suspension cultures of Dorotheanthus bellidiformis (Burm. f.) N.E.Br.. Planta 199, 244–250 (1996). https://doi.org/10.1007/BF00196565
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00196565