title: Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase.
abstract: Dethiobiotin synthetase (DTBS) plays a crucial role in biotin biosynthesis in microorganisms, fungi, and plants. Due to its importance in bacterial pathogenesis, and the absence of a human homologue, DTBS is a promising target for the development of new antibacterials desperately needed to combat antibiotic resistance. Here we report the first X-ray structure of DTBS from Mycobacterium tuberculosis (MtDTBS) bound to a nucleotide triphosphate (CTP). The nucleoside base is stabilized in its pocket through hydrogen-bonding interactions with the protein backbone, rather than amino acid side chains. This resulted in the unexpected finding that MtDTBS could utilise ATP, CTP, GTP, ITP, TTP, or UTP with similar Km and kcat values, although the enzyme had the highest affinity for CTP in competitive binding and surface plasmon resonance assays. This is in contrast to other DTBS homologues that preferentially bind ATP primarily through hydrogen-bonds between the purine base and the carboxamide side chain of a key asparagine. Mutational analysis performed alongside in silico experiments revealed a gate-keeper role for Asn175 in Escherichia coli DTBS that excludes binding of other nucleotide triphosphates. Here we provide evidence to show that MtDTBS has a broad nucleotide specificity due to the absence of the gate-keeper residue.
authors: School of Biological Sciences, The University of Adelaide, South Australia, 5005, Australia.
authors: Wanisa
authors: W
authors: Salaemae
authors: Department of Biochemistry and Molecular Biology, School of Biomedical Science, Monash University, Victoria, 3800, Australia.
authors: Min Y
authors: MY
authors: Yap
authors: School of Biological Sciences, The University of Adelaide, South Australia, 5005, Australia.
authors: Kate L
authors: KL
authors: Wegener
authors: School of Biological Sciences, The University of Adelaide, South Australia, 5005, Australia.
authors: Grant W
authors: GW
authors: Booker
authors: Department of Biochemistry and Molecular Biology, School of Biomedical Science, Monash University, Victoria, 3800, Australia.
authors: Matthew C J
authors: MC
authors: Wilce
authors: School of Biological Sciences, The University of Adelaide, South Australia, 5005, Australia. Electronic address: steven.polyak@adelaide.edu.au.
authors: Steven W
authors: SW
authors: Polyak
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009711:Nucleotides; D019731:Carbon-Nitrogen Ligases; C020024:dethiobiotin synthetase
keywords: Biotin biosynthesis; Crystal structure; Dethiobiotin synthetase; Enzyme; Escherichia coli; Mycobacterium tuberculosis; Nucleotide triphosphate