Substrate binding and carboxylation by dethiobiotin synthetase--a kinetic and X-ray study.
BACKGROUND
The vitamin biotin is a ubiquitous prosthetic group of carboxylase and transcarboxylase enzymes. Biotin biosynthesis occurs by similar pathways in microorganisms and plants. The penultimate step in biotin biosynthesis, catalyzed by dethiobiotin synthetase (DTBS), involves a unique ATP-dependent N-carboxylation, resulting in formation of the ureido ring function of dethiobiotin. The first two steps of dethiobiotin formation, which is a complex, multistep enzymatic reaction, have been elucidated by a combination of X-ray crystallography and kinetic methods.


RESULTS
The first step in catalysis by DTBS is the formation of an enzyme-substrate complex and the second is the enzymatic carboxylation of the bound substrate. Both steps are Mg2+ dependent. The kinetic constants in the presence and absence of Mg2+ have been measured and a set of X-ray structures determined at different stages of the reaction. The conformational changes in the active site of the enzyme, induced by Mg2+, substrate binding and substrate carboxylation, have been monitored crystallographically and are discussed. Sulfate ions bound to DTBS may mimic the behaviour of the alpha- and gamma-phosphates of ATP in Mg2+ binding and in the subsequent steps of the reaction.


CONCLUSIONS
Mg2+ is an essential cation for both substrate binding and carbamate formation by DTBS, when sulfate is present. The conformational changes induced at the active site in the DTBS-substrate complex, when Mg2+ is present, are small yet highly significant and serve to optimize the interactions between substrate and enzyme. DTBS is active as a homodimer and the substrate-binding site straddles both monomers in the dimer. The carboxylation site is unambiguously identified as the N-7 amino group of the substrate, rather than the N-8 amino group, as previously suggested. The elongated nucleotide-binding loop (the P loop) binds both ATP and substrate in a manner which suggests that this feature may be of wider importance.
Alexeev
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Department of Biochemistry, University of Edinburgh, UK.
Baxter
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RL
Smekal
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Sawyer
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D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D000599:Amino Acids, Diamino / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D001710:Biotin / Q000096:biosynthesis; D019731:Carbon-Nitrogen Ligases; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008025:Ligases / Q000737:chemistry*; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D013431:Sulfates / Q000378:metabolism
D000599:Amino Acids, Diamino; D001426:Bacterial Proteins; D013431:Sulfates; C004720:7,8-diaminopelargonic acid; D001710:Biotin; D000255:Adenosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C020024:dethiobiotin synthetase; D008274:Magnesium