TY - JOUR

T1 - Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase

AU - Thymark, Majbritt

AU - Johansson, Eva

AU - Larsen, Sine

AU - Willemoës, Martin

N1 - Keywords: Binding Sites; Computer Simulation; Enzyme Activation; Escherichia coli; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Nucleotide Deaminases; Protein Binding

PY - 2007

Y1 - 2007

N2 - In Escherichia coli and Salmonella typhimurium about 80% of the dUMP used for dTMP synthesis is derived from deamination of dCTP. The dCTP deaminase produces dUTP that subsequently is hydrolyzed by dUTPase to dUMP and diphosphate. The dCTP deaminase is regulated by dTTP that inhibits the enzyme by binding to the active site and induces an inactive conformation of the trimeric enzyme. We have analyzed the role of residues previously suggested to play a role in catalysis. The mutant enzymes R115Q, S111C, S111T and E138D were all purified and analyzed for activity. Only S111T and E138D displayed detectable activity with a 30- and 140-fold reduction in k(cat), respectively. Furthermore, S111T and E138D both showed altered dTTP inhibition compared to wild-type enzyme. S111T was almost insensitive to the presence of dTTP. With the E138D enzyme the dTTP dependent increase in cooperativity of dCTP saturation was absent, although the dTTP inhibition itself was still cooperative. Modeling of the active site of the S111T enzyme indicated that this enzyme is restricted in forming the inactive dTTP binding conformer due to steric hindrance by the additional methyl group in threonine. The crystal structure of E138D in complex with dUTP showed a hydrogen bonding network in the active site similar to wild-type enzyme. However, changes in the hydrogen bond lengths between the carboxylate and a catalytic water molecule as well as a slightly different orientation of the pyrimidine ring of the bound nucleotide may provide an explanation for the reduced activity.

AB - In Escherichia coli and Salmonella typhimurium about 80% of the dUMP used for dTMP synthesis is derived from deamination of dCTP. The dCTP deaminase produces dUTP that subsequently is hydrolyzed by dUTPase to dUMP and diphosphate. The dCTP deaminase is regulated by dTTP that inhibits the enzyme by binding to the active site and induces an inactive conformation of the trimeric enzyme. We have analyzed the role of residues previously suggested to play a role in catalysis. The mutant enzymes R115Q, S111C, S111T and E138D were all purified and analyzed for activity. Only S111T and E138D displayed detectable activity with a 30- and 140-fold reduction in k(cat), respectively. Furthermore, S111T and E138D both showed altered dTTP inhibition compared to wild-type enzyme. S111T was almost insensitive to the presence of dTTP. With the E138D enzyme the dTTP dependent increase in cooperativity of dCTP saturation was absent, although the dTTP inhibition itself was still cooperative. Modeling of the active site of the S111T enzyme indicated that this enzyme is restricted in forming the inactive dTTP binding conformer due to steric hindrance by the additional methyl group in threonine. The crystal structure of E138D in complex with dUTP showed a hydrogen bonding network in the active site similar to wild-type enzyme. However, changes in the hydrogen bond lengths between the carboxylate and a catalytic water molecule as well as a slightly different orientation of the pyrimidine ring of the bound nucleotide may provide an explanation for the reduced activity.

U2 - 10.1016/j.abb.2007.10.013

DO - 10.1016/j.abb.2007.10.013

M3 - Journal article

C2 - 17996716

VL - 470

SP - 20

EP - 26

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1

ER -