1xs4: Difference between revisions

Revision as of 16:58, 21 February 2008

dCTP deaminase from Escherichia coli- E138A mutant enzyme in complex with dCTP

OverviewOverview

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.

About this StructureAbout this Structure

1XS4 is a Single protein structure of sequence from Escherichia coli with and as ligands. Active as dCTP deaminase, with EC number 3.5.4.13 Full crystallographic information is available from OCA.

ReferenceReference

Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes., Johansson E, Fano M, Bynck JH, Neuhard J, Larsen S, Sigurskjold BW, Christensen U, Willemoes M, J Biol Chem. 2005 Jan 28;280(4):3051-9. Epub 2004 Nov 10. PMID:15539408

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-[[Image:1xs4.gif|left|200px]]<br /><applet load="1xs4" size="450" color="white" frame="true" align="right" spinBox="true"
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 '''dCTP deaminase from Escherichia coli- E138A mutant enzyme in complex with dCTP'''<br />
 ==Overview==
-dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming, dUTP that via dUTPase is the main pathway providing substrate for, thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP, deaminase is unique among nucleoside and nucleotide deaminases as it, functions without aid from a catalytic metal ion that facilitates, preparation of a water molecule for nucleophilic attack on the substrate., Two active site amino acid residues, Arg(115) and Glu(138), were, identified by mutational analysis as important for activity in E. coli, dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any, detectable activity but circular dichroism spectra for all mutant enzymes, were similar to wild type suggesting that the overall structure was not, changed. The crystal structures of wild-type E. coli dCTP deaminase and, the E138A mutant enzyme have been determined in complex with dUTP and, Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The, enzyme is a third member of the family of the structurally related, trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from, Methanocaldococcus jannaschii. However, the C-terminal fold is completely, different from dUTPases resulting in an active site built from residues, from two of the trimer subunits, and not from three subunits as in, dUTPases. The nucleotides are well defined as well as Mg(2+) that is, tridentately coordinated to the nucleotide phosphate chains. We suggest a, catalytic mechanism for the dCTP deaminase and identify structural, differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.
+dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.
 ==About this Structure==
-XS4 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with MG and DCP as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/dCTP_deaminase dCTP deaminase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.4.13 3.5.4.13] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1XS4 OCA].
+XS4 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=MG:'>MG</scene> and <scene name='pdbligand=DCP:'>DCP</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/dCTP_deaminase dCTP deaminase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.4.13 3.5.4.13] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1XS4 OCA].
 ==Reference==
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 [[Category: Single protein]]
 [[Category: dCTP deaminase]]
-[[Category: Bynck, J.H.]]
+[[Category: Bynck, J H.]]
 [[Category: Christensen, U.]]
 [[Category: Fano, M.]]
@@ Line 20: / Line 20: @@
 [[Category: Larsen, S.]]
 [[Category: Neuhard, J.]]
-[[Category: Sigurskjold, B.W.]]
+[[Category: Sigurskjold, B W.]]
 [[Category: Willemoes, M.]]
 [[Category: DCP]]
@@ Line 28: / Line 28: @@
 [[Category: trimer]]
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