Aspects related to mevalonate biosynthesis in plants.
We purified and characterized a membrane-associated enzyme system from radish (Raphanus sativus L.) that is capable of converting acetyl-CoA into 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA). The enzyme system apparently comprises acetoacetyl-CoA thiolase (EC 2.3.1.9) and HMG-CoA synthase (EC 4.1.3.5). Its activity in vitro can be strongly stimulated by FeII. When ferrous ions are applied chelated with ethylenediaminetetraacetate, citrate or adenosine 5-triphosphate (ATP), the stimulation is further increased. Stimulation is due to a higher catalytic efficiency as indicated by an increase in Vmax, whereas the affinity of the enzyme towards acetyl-CoA remains constant (Km = 6 micro M). A considerable portion of HMG-CoA lyase activity is associated with the same membranes. HMG-CoA lyase (EC 4.1.3.4) is also solubilized and partially co-purified. Its activity requires comparatively high concentrations of Mg2+. The conversion of HMG-CoA to mevalonic acid is catalyzed by HMG-CoA reductase (EC 1.1.1.34) that is associated with the same membranes. By cDNA encoding the Arabidopsis HMG-CoA reductase, we isolated a corresponding gene from a cDNA library newly established from etiolated radish seedlings. This full-length cDNA, referred to as lambda cRS3, encodes a polypeptide 583 amino acids with a molecular mass of about 63 kDa. The hydropathy profile suggests the presence of two hydrophobic membrane-spanning domains within the N-terminal 165 amino acids. The carboxy-terminal part, where the catalytic site resides, is highly conserved in all eukaryotic HMG-CoA reductase genes sequenced so far.
Botanisches Institut, Universität Karlsruhe, Germany.
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Bach
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Boronat
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Caelles
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Ferrer
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Weber
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Wettstein
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000101:Acetyl-CoA C-Acetyltransferase / Q000302:isolation & purification / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000302:isolation & purification / Q000378:metabolism; D008798:Mevalonic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000378:metabolism; D010944:Plants / Q000201:enzymology*
D000214:Acyl Coenzyme A; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D000105:Acetyl Coenzyme A; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases; D000101:Acetyl-CoA C-Acetyltransferase; D006904:Hydroxymethylglutaryl-CoA Synthase; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D008798:Mevalonic Acid