Fig. 2. Intermediates and enzymes involved in the first steps of terpenoid biosynthesis. HMG-CoA lyase and MG-CoA hydratase indicate branching (competitive ?) pathways.
25°C and pH 8.1). The spectrophotometric assay is based on the monitoring of the, CoASH depending, rate of consumption of acetoacetyl-CoA at 300 nm. In crude extracts of suspension cultured C. roseus cells, typically a specific activity of about 1 nkat/mg protein is found. In the intact plant highest activities were found in the flowers and stems, 1.1 and 1.6 nkat/mg protein respectively . The enzyme was purified by affinity (Fractogel TSK-AF Orange) and anion-exchange (Mono Q) chromatography . After gel filtration and SDS-PAGE it was found that the enzyme is a tetrameric enzyme consisting of identical subunits of 41 kDa. This is the first report on the purification of this thiolase from a plant source, as far as characterized its physical and kinetic properties are similar to the thiolase from yeast and mammalian cells.
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (EC 18.104.22.168) couples another acetyl-CoA molecule to acetoacetyl-CoA, yielding HMG-CoA and CoASH. The reaction is essentially irreversible. In cell cultures of C. roseus the specific activity of the enzyme is typically 75 nkat/mg protein. Flowers and stems showed slightly higher activities, 130 and 120 nkat/mg respectively . The unstable enzyme was partially purified from C. roseus suspension cultured cells by ionexchange chromatography and gel filtration . The enzyme is irreversibly inhibited by L-659,699, a metabolite known to inhibit mammalian HMG-CoA synthase specifically, indicating a similar catalytic mechanism for the plant enzyme.
Mevalonate kinase (EC 22.214.171.124) phosphorylates mevalonic acid, the NADPH-reduced form of HMG-CoA. The reaction is ATP and Mg2+ dependent. The enzyme was purified from a C. roseus cell suspension culture, in which, after induction a specific activity of 1.5 nkat/mg protein is found , The purification protocol comprised ion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. By gel filtration an Mr of 105,000 was found for mevalonate kinase.
Isopentenyl diphosphate (IPP) isomerase (EC 126.96.36.199) catalyses the interconversion of IPP and dimethylallyl diphosphate (DMAPP). During a growth cycle of a suspension culture of C. roseus, relatively highest IPP isomerase activity was found between 4 and 6 days after subculture: about 20 pkat/mg protein. By Western blot analysis, using purified polyclonal antibodies raised against IPP isomerase from Capsicum annuum chloroplasts, two isoforms of IPP isomerase were detected in the C. roseus extracts .
DMAPP is a central intermediate in isoprenoid biosynthesis (fig. 1). It may be used as a starter molecule for the formation of higher isoprenoids (C10, C15, C30, etc.) or it can be used for prenylation of (aromatic) precursors in secondary metabolism, for example as found in anthraquinones. We studied the role of IPP
isomerase in the elicitor-inducible formation of anthraquinones in cell cultures of Cinchona robusta. Typically, IPP isomerase activity found in untreated cells is 15 pkat/mg protein. Twelve hours after induction this activity is (transiently) increased to 40 pkat/mg protein. Two isoforms of IPP isomerase, differing in molecular weight and cofactor requirement, were purified. One isoform was specifically induced after elicitation .
HMG-CoA lyase (EC 188.8.131.52) catalyses the formation of acetoacetate and acetyl-CoA from HMG-CoA. This enzyme is not directly involved in isoprenoid biosynthesis, but it may compete for the substrate HMG-CoA. In mammalian cells this enzyme is involved in the formation of ketone bodies, a process not described for plants so far . For this enzyme a novel HPLC assay was developed (manuscript in preparation), which has been used for a screening of several plant cell cultures. In C. roseus cell cultures a specific activity of 20-40 pkat/mg protein was found. In other cultures the enzyme showed also to be present, activities between 20-90 pkat/mg protein were found for among others Rubia, Morinda and Tabernaemontana cultures. The unstable C. roseus enzyme was partially purified from suspension cultured cells, maximum activity was found at pH 8.2 in the presence of 20 mM Mg2+ and 5 mM dithiothreitol.
3-Methylglutaconyl-CoA (MG-CoA) hydratase (EC 184.108.40.206) activity yields MG-CoA from the substrate HMG-CoA. As for HMG-CoA lyase, this enzyme may compete with terpenoid biosynthesis. In mammalian cells this enzyme is involved in leucine metabolism. The activity of the enzyme was detected in various plant cell cultures, among others Arabidopsis thaliana. The C. roseus enzyme was partially purified by ammonium sulfate precipitation, ion- exchange and hydrcxyapatite chromatography, and gel filtration . It is relatively stable and does not require cofactors.
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