Katharine J. Gibson

Crystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 å resolution

BACKGROUND In Escherichia coli, the enzymes of the biotin biosynthesis pathway are encoded by the bio operon. One of these enzymes, ATP-dependent dethiobiotin synthetase, catalyzes the carboxylation of 7,8-diaminopelargonic acid leading to the formation of the ureido ring of biotin. The enzyme belongs to the class of ATP-dependent carboxylases and we present here the first crystal structure determined for this class of enzyme. RESULTS We have determined the crystal structure of homodimeric dethiobiotin synthetase to 1.65 A resolution. The subunit consists of a seven-stranded parallel beta-sheet, surrounded by alpha-helices. The sheet contains the classical mononucleotide-binding motif with a fingerprint peptide Gly-X-X-X-X-X-Gly-Lys-Thr. The mononucleotide binding part of the structure is very similar to the GTP-binding protein H-ras-p21 and thus all GTP-binding proteins. A comparison reveals that some of the residues, which in H-ras-p21 interact with the nucleotide and the metal ion, are conserved in the synthetase. CONCLUSIONS The three-dimensional structure of dethiobiotin synthetase has revealed that ATP-dependent carboxylases contain the classical mononucleotide-binding fold. Considerable similarities to the structure of the GTP-binding protein H-ras-p21 were found, indicating that both proteins might have evolved from a common ancestral mononucleotide-binding fold.

Purification and preliminary X-ray crystallographic studies of recombinant 7,8-diaminopelargonic acid synthase from Escherichia coli

Recombinant 7,8-diaminopelargonic acid synthase from Escherichia coli, a pyridoxal-phosphate-dependent aminotransferase, has been crystallized in space groups P21 and C2. Both crystal forms were obtained at pH 7.3 with 21% polyethylene glycol and 10% 2-propanol as precipitants. The cell dimensions were a = 130, b = 57.5, c = 117 A, beta = 110 degrees for the C2 crystals, and a = 58.4, b = 55.6, c = 121 A, beta = 96.9 degrees for the P21 crystals, which diffract to at least 2.6 and 2.0 A resolution, respectively.