STRUCTURE OF THE BIOTINYL DOMAIN OF ACETYL-COENZYME-A CARBOXYLASE DETERMINED BY MAD PHASING

Background: Acetyl-coenzyme A carboxylase catalyzes the first committe d step of fatty acid biosynthesis. Universally, this reaction involves three functional components all related to a carboxybiotinyl intermed iate. A biotinyl domain shuttles its covalently attached biotin prosth etic group between the active sites of a biotin carboxylase and a carb oxyl transferase. In Escherichia coli, the three components reside in separate subunits; a biotinyl domain is the functional portion of one of these, biotin carboxyl carrier protein (BCCP). Results: We have exp ressed natural and selenomethionyl (Se-met) BCCP from E. coli as bioti nylated recombinant proteins, proteolyzed them with subtilisin Carlsbe rg to produce the biotinyl domains BCCPSC and Se-met BCCPSC, determine d the crystal structure of Se-met BCCPSC using a modified version of t he multiwavelength anomalous diffraction (MAD) phasing protocol, and r efined the structure for the natural BCCPSC at 1.8 Angstrom resolution . The structure may be described as a capped beta sandwich with quasi- dyad symmetry. Each half contains a characteristic hammerhead motif. T he biotinylated lysine is located at a hairpin beta turn which connect s the two symmetric halves of the molecule, and its biotinyl group int eracts with a non-symmetric protrusion from the core. Conclusions: Thi s first crystal structure of a biotinyl domain helps to unravel the ce ntral role of such domains in reactions catalyzed by biotin-dependent carboxylases. The hammerhead structure observed twice in BCCPSC may be regarded as the basic structural motif of biotinyl and lipoyl domains of a superfamily of enzymes. The new MAD phasing techniques developed in the course of determining this structure enhance the power of the MAD method.