The catalytic site of monogalactosyldiacylglycerol synthase from spinach chloroplast envelope membranes. Biochemical analysis of the structure and of the metal content.
We have analyzed the structure of the active site of monogalactosyldiacylglycerol (MGDG) synthase from spinach chloroplast envelope. Since purification of this membrane-embedded enzyme yielded such low amounts of protein that analyses of the amino acid sequence were so far impossible, we used indirect strategies. Analyses of the inhibition of MGDG synthase by UDP and of its inactivation by citraconic anhydride first indicated that the enzyme contained two functionally independent and topologically distinct binding sites for each substrate. Whereas MGDG synthase binds both the nucleotidic part of UDP-Gal and the acyl chains of 1,2-diacylglycerol, UDP is a competitive inhibitor relatively to UDP-Gal, while it does not compete with 1,2-diacylglycerol for binding on the enzyme. The UDP-Gal-binding site contains lysine residues, as demonstrated for UDP-Gal-binding sites from all galactosyltransferases studied so far. Radiolabeling of MGDG synthase by sulfur labeling reagent, a 35S-labeled lysine-blocking reagent, confirmed that MGDG synthase was a polypeptide with a low molecular mass (around 20 kDa). The 1,2-diacylglycerol-binding site contains reduced cysteines and at least one metal. The divalent cation(s) associated to apo-MGDG synthase was not unambiguously identified, but the results suggest that it could be zinc. Therefore, MGDG synthase presents some structural features in common with diacylglycerol-manipulating enzymes, such as protein kinase C and 1,2-diacylglycerol kinase, which are characterized by the presence of a ubiquitous Cys6His2 domain involved in zinc coordination in their 1,2-diacylglycerol-binding domains.
Maréchal
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Département de Biologie Moleculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, France.
Miège
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C
Block
M A
MA
Douce
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Joyard
J
J
D001665:Binding Sites; D002413:Cations, Divalent / Q000032:analysis* / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D002736:Chloroplasts / Q000201:enzymology*; D003545:Cysteine / Q000032:analysis; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005700:Galactosyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine / Q000032:analysis; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D018724:Spinacia oleracea / Q000201:enzymology*; D013462:Sulfur Radioisotopes; D014530:Uridine Diphosphate / Q000494:pharmacology; D015032:Zinc / Q000032:analysis; D017967:Zinc Compounds / Q000494:pharmacology
D002413:Cations, Divalent; D002614:Chelating Agents; D002712:Chlorides; D013462:Sulfur Radioisotopes; D017967:Zinc Compounds; D006639:Histidine; D014530:Uridine Diphosphate; C016837:zinc chloride; D005700:Galactosyltransferases; C109990:1,2-diacylglycerol 3-beta-galactosyltransferase; D015032:Zinc; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol