Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing.
BACKGROUND
Acetyl-coenzyme A carboxylase catalyzes the first committed step of fatty acid biosynthesis. Universally, this reaction involves three functional components all related to a carboxybiotinyl intermediate. A biotinyl domain shuttles its covalently attached biotin prosthetic group between the active sites of a biotin carboxylase and a carboxyl transferase. In Escherichia coli, the three components reside in separate subunits: a biotinyl domain is the functional portion of one of these, biotin carboxy carrier protein (BCCP).


RESULTS
We have expressed natural and selenomethionyl (Se-met) BCCP from E. coli as biotinylated recombinant proteins, proteolyzed them with subtilisin Carlsberg to produce the biotinyl domains BCCP and Se-met BCCPsc, determined the crystal structure of Se-met BCCPsc using a modified version of the multiwavelength anomalous diffraction (MAD) phasing protocol, and refined the structure for the natural BCCPsc at 1.8 A resolution. The structure may be described as a capped beta sandwich with quasi-dyad symmetry. Each half contains a characteristic hammerhead motif. The biotinylated lysin is located at a hairpin beta turn which connects the two symmetric halves of the molecule, and its biotinyl group interacts with a non-symmetric protrusion from the core.


CONCLUSIONS
This first crystal structure of a biotinyl domain helps to unravel the central 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.
Athappilly
F K
FK
Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA.
Hendrickson
W A
WA
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry* / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D019143:Evolution, Molecular; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D050841:Glycine Dehydrogenase (Decarboxylating); D006801:Humans; D006860:Hydrogen Bonding; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009100:Multiple Carboxylase Deficiency / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship
C099010:BCCPsc protein, recombinant; D001426:Bacterial Proteins; D010446:Peptide Fragments; D010940:Plant Proteins; D011768:Pyruvate Dehydrogenase Complex; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C099011:Se-Met-BCCPsc protein, recombinant; D001710:Biotin; D000594:Amino Acid Oxidoreductases; D050841:Glycine Dehydrogenase (Decarboxylating); D000103:Acetyl-CoA Carboxylase; C013411:biocytin; D008239:Lysine