Mechanistic implications and family relationships from the structure of dethiobiotin synthetase.
BACKGROUND
Biotin is the vitamin essential for many biological carboxylation reactions, such as the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA in fatty acid biosynthesis. Dethiobiotin synthetase (DTBS) facilitates the penultimate, ureido ring closure in biotin synthesis, which is a non-biotin-dependent carboxylation. DTBS displays no sequence similarity to any other protein in the database. Structural studies provide a molecular insight into the reaction mechanism of DTBS.


RESULTS
We present the structure of DTBS refined to 1.80 A resolution with an R-factor of 17.2% for all terms plus unrefined data on the binding of the substrate, 7,8-diaminopelargonic acid and the product, dethiobiotin. These studies confirm that the protein forms a homodimer with each subunit folded as a single globular alpha/beta domain. The presence of sulphate ions in the crystals and comparisons with the related Ha-ras-p21 oncogene product are used to infer the ATP-binding site, corroborated by the difference electron density for the ATP analogue AMP-PNP.


CONCLUSIONS
This study establishes that the enzyme active site is situated at the dimer interface, with the substrate binding to one monomer and ATP to the other. The overall fold of DTBS closely resembles that of three other enzymes, adenylosuccinate synthetase (purA), Ha-ras-p21, and nitrogenase iron protein, that are unrelated by sequence or function, indicating that DTBS is a member of a diverse family of enzymes.
Alexeev
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Edinburgh Centre for Molecular Recognition, Department of Biochemistry, University of Edinburgh, UK.
Baxter
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RL
Sawyer
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D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000599:Amino Acids, Diamino; D001665:Binding Sites / Q000235:genetics; D019731:Carbon-Nitrogen Ligases; D018360:Crystallography, X-Ray; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008025:Ligases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000599:Amino Acids, Diamino; C004720:7,8-diaminopelargonic acid; D000255:Adenosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C020024:dethiobiotin synthetase