Mycobacterium tuberculosis Dethiobiotin Synthetase Facilitates Nucleoside Triphosphate Promiscuity through Alternate Binding Modes
Date
2018
Authors
Thompson, A.P.
Salaemae, W.
Pederick, J.L.
Abell, A.D.
Booker, G.W.
Bruning, J.B.
Wegener, K.L.
Polyak, S.W.
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Journal article
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ACS Catalysis, 2018; 8(11):10774-10783
Statement of Responsibility
Andrew P. Thompson, Wanisa Salaemae, Jordan L. Pederick, Andrew D. Abell, Grant W. Booker, John B. Bruning, Kate L. Wegener and Steven W. Polyak
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Abstract
The penultimate step in the biosynthesis of biotin is the closure of the ureido heterocycle in a reaction requiring a nucleoside triphosphate (NTP). In Mycobacterium tuberculosis this reaction is catalyzed by dethiobiotin synthetase (MtDTBS). MtDTBS is unusual as it can employ multiple (NTPs), with a >100-fold preference for cytidine triphosphate (CTP). Here the molecular basis of NTP binding was investigated using a surface plasmon resonance-based ligand binding assay and X-ray crystallography. The biophysical and structural data revealed two discrete mechanisms by which MtDTBS binds NTPs: (i) A high affinity binding mode employed by CTP (KD 160 nM) that is characterized by a slow dissociation rate between enzyme and ligand (kd 5.3 × 10–2 s–1) and that is defined by an extended network of specific ligand–protein interactions involving both the cytidine and triphosphate moieties and (ii) a low affinity mode employed by the remaining NTPs (KD > 16.5 μM), that is characterized by weak interactions between protein and ligand. Previously intractable structures of MtDTBS in complex with ATP, GTP, UTP, and ITP were obtained to define the molecular basis of the low affinity ligand binding. Anchoring of the triphosphate moiety into the phosphate binding loop of MtDTBS allows the promiscuous utilization of multiple NTPs. Both high and low binding mechanisms showed conserved hydrogen bonding interactions involving the β-phosphate of NTPs and a high-affinity anion binding site within the phosphate binding loop. This study provides insights into enzymes that can likewise utilize multiple NTPs.
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© 2018 American Chemical Society.