| Abstract
| - 1,2-Diacylglycerol 3-glucosyltransferase is associated with the membrane surface catalyzingthe synthesis of the major nonbilayer-prone lipid α-monoglucosyl diacylglycerol (MGlcDAG) from 1,2-DAG in the cell wall-less Acholeplasma laidlawii. Phosphatidylglycerol (PG), but not neutral or zwitterioniclipids, seems to be essential for an active conformation and function of the enzyme. Surface plasmonresonance analysis was employed to study association of the enzyme with lipid bilayers. Binding kineticscould be well fitted only to a two-state model, implying also a (second) conformational step. The enzymebound less efficiently to liposomes containing only zwitterionic lipids, whereas increasing molar fractionsof the anionic PG or cardiolipin (CL) strongly promoted binding by improved association (ka1), andespecially a decreased rate of return (kd2) from the second state. This yielded a very low overall dissociationconstant (KD), corresponding to an essentially irreversible membrane association. Both liposome bindingand consecutive activity of the enzyme correlated with the PG concentration. The importance of theelectrostatic interactions with anionic lipids was shown by quenching of both binding and activity withincreasing NaCl concentrations, and corroborated in vivo for an active enzyme−green fluorescent proteinhybrid in Escherichia coli. Nonbilayer-prone lipids substantially enhanced enzyme−liposome binding bypromoting a changed conformation (decreasing kd2), similar to the anionic lipids, indicating the importanceof hydrophobic interactions and a curvature packing stress. For CL and the nonbilayer lipids, effects onenzyme binding and consecutive activity were not correlated, suggesting a separate lipid control of activity.Similar features were recorded with polylysine (cationic) and polyglutamate (anionic) peptides present,but here probably dependent on the selective charge interactions with the enzyme N- and C-domains,respectively. A lipid-dependent conformational change and PG association of the enzyme were verifiedby circular dichroism, intrinsic tryptophan, and pyrene-probe fluorescence analyses, respectively. It isconcluded that an electrostatic association of the enzyme with the membrane surface is accompanied byhydrophobic interactions and a conformational change. However, specific lipids, the curvature packingstress, and proteins or small molecules bound to the enzyme can modulate the activity of the bound A.laidlawii MGlcDAG synthase.
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