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 monogalactosyldia cylglycerol (MGDG) synthase from spinach chloroplast envelope. Since p urification of this membrane-embedded enzyme yielded such low amounts of protein that analyses of the amino acid sequence were so far imposs ible, 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 t opologically distinct binding sites for each substrate, Whereas MGDG s ynthase binds both the nucleotidic part of UDP-Gal and the acyl chains of 1,2-diacylglycerol, UDP is a competitive inhibitor relatively to U DP-Gal, while it does not compete with 1,2-diacylglycerol for binding on the enzyme, The UDP-Gal-binding site contains lysine residues, as d emonstrated for UDP-Gal-binding sites from all galactosyltransferases studied so far, Radiolabeling of MGDG synthase by sulfur labeling reag ent, a S-35-labeled lysine-blocking reagent, confirmed that MGDG synth ase 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 b e zinc, Therefore, MGDG synthase presents some structural features in common with diacylglycerol-manipulating enzymes, such as protein kinas e C and 1,2-diacylglycerol kinase, which are characterized by the pres ence of a ubiquitous Cys(6)His(2) domain involved in zinc coordination in their 1,2-diacylglycerol-binding domains.