Structure
Research ArticleCrystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 å resolution
Introduction
Biotin (vitamin H) is a cofactor in many biological carboxylation reactions, but is present only in very small amounts in mammalian cells. The vitamin is synthesized in plants and some microorganisms. In Escherichia coli, the necessary enzymes of biotin biosynthesis are encoded in a gene cluster, the bio operon. This cluster consists of five genes, bioA, B, F, C and D [1]. The nucleotide sequence of the bio operon has been determined [2]. These genes encode five different enzymes, which are involved in the synthesis of biotin from pimeloyl-coenzyme A.
Dethiobiotin synthetase (DTBS) catalyzes the penultimate step in biotin biosynthesis, the formation of the ureido ring of biotin, converting 7,8-diaminopelargonic acid to dethiobiotin (Figure 1). DTBS is a homodimer with a molecular weight of 24 kDa per subunit [3]. The enzyme is mechanistically of considerable interest, since it is an example of a carboxylase which does not use biotin as a coenzyme. Its mechanism is also different from the carboxylation reaction catalyzed by ribulose bisphosphate carboxylase in the fixation of CO 2.
DTBS uses CO 2 as second substrate and requires Mg-ATP as a cofactor. The proposed reaction pathway involves formation of a carbamate with one of the amines of the substrate which then reacts with Mg-ATP to give the mixed phosphoric-carbamic acid anhydride plus Mg-ADP. The mixed anhydride is then thought to react with the other amino group of the substrate to yield dethiobiotin and phosphate.
As the first step in a biochemical and structural characterization of the biotin biosynthesis pathway, we have crystallized DTBS from E. coli and determined its three- dimensional structure by protein crystallography. In this paper, we describe the refined three-dimensional structure of the enzyme which reveals the fold for an ATP-dependent carboxylase, at 1.65 å resolution.
Section snippets
Crystallization and structure determination
The crystals of DTBS are monoclinic, space group C2 with cell dimensions a = 73.2 å, b = 49.2 å and c = 61.8 å, β = 107.1° . A very similar crystal form has been reported recently using ammonium sulfate as precipitant [4]. The crystals are stable in the X-ray beam and diffract to very high resolution. On a conventional rotating anode, diffraction is observed to at least 1.65 å resolution. Based on a molecular weight of 24 kDa ([4], and Lorimer and Miller, unpublished data), the packing density
Biological implications
Biological carboxylation reactions are catalyzed by several classes of enzymes with different mechanisms. These classes include biotin-dependent enzymes, ribulose bisphosphate carboxylase, and the ATP-dependent carboxylases such as dethiobiotin synthetase (DTBS).
We have determined the crystal structure of DTBS. As this is the first structure of an ATP -dependent carboxylase, it reveals the fold of this class of enzyme. The enzyme is a homodimer, each monomer consisting of a seven-stranded
Acknowledgements
We thank Prof. Gerry Cohen, University of Tel Aviv for the gift of an overexpressing strain of E. coli. We also thank the following individuals for their contributions to the determination of the correct primary structure: Dr. Howard Hershey and Linda Besl for nucleotide sequencing, Tom Miller, Tom Webb and Pat Webber for amino acid sequencing and Dr. Barbara Larsen for electrospray MS.
Weijun Huang, Ylva Lindqvist and Gunter Schneider (corresponding author), Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center Box 590, S-751 24 Uppsala, Sweden.
Katharine J Gibson, Dennis Flint and George Lorimer, DuPont Central Research and Development, Experimental Station, PO Box 80402, Wilmington, DE 19880-0402, USA.
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Weijun Huang, Ylva Lindqvist and Gunter Schneider (corresponding author), Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center Box 590, S-751 24 Uppsala, Sweden.
Katharine J Gibson, Dennis Flint and George Lorimer, DuPont Central Research and Development, Experimental Station, PO Box 80402, Wilmington, DE 19880-0402, USA.