Cloning, purification, and characterization of the Shigella boydii dGTP triphosphohydrolase.
The nucleotide sequence of the Shigella boydii dgt gene, which encodes the enzyme deoxyguanosine triphosphate triphosphohydrolase (dGTPase, EC 3.1.5.1), has been determined. The 1515-nucleotide Shigella dgt open reading frame has been subcloned into a T7 RNA polymerase-based expression vector. The resulting expressed protein has been purified to homogeneity using a novel single-day chromatographic regime. The protocol includes ion exchange, affinity, and hydrophobic interaction chromatography. The purified 505-amino acid (59.4 kDa) protein exists in solution as a heat-stable homotetramer, and enzymatic assays reveal that the expressed enzyme is fully active. Substrate specificity can be explained by the array of potential hydrogen bond donors/acceptors displayed on the base moiety of the (deoxy)nucleoside triphosphate. Shigella dGTPase can be inhibited by the addition of stoichiometric amounts of reducing agents. The loss of activity is both time- and concentration-dependent and is accompanied by a decrease in the thermal stability of the enzyme. Shigella dGTPase in the fully reduced form is destabilized by 1.8 kcal/mol compared with the oxidized form. Hence, disulfide bonds play a pivotal role in the maintenance of dGTPase stability and enzymatic functionality. Initial Shigella dGTPase protein crystals have been formed.
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School of Chemistry and Biochemistry, The Georgia Institute of Technology, Atlanta 30332, USA. stephen.quirk@chemistry.gatech.edu
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D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004229:Dithiothreitol / Q000737:chemistry; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins; D012761:Shigella boydii / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010744:Phosphoric Monoester Hydrolases; D004229:Dithiothreitol