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Tying down the arm in Bacillus dUTPase: structure and mechanism

Homotrimeric dUTPases contain three active sites, each formed by five conserved sequence motifs originating from all three subunits. The essential fifth motif lies in a flexible C‐terminal arm which becomes ordered during catalysis and is disordered in most crystal structures. Previously, it has bee...

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Published in:Acta crystallographica. Section D, Biological crystallography. Biological crystallography., 2013-08, Vol.69 (8), p.1367-1380
Main Authors: García-Nafría, Javier, Timm, Jennifer, Harrison, Charlotte, Turkenburg, Johan P., Wilson, Keith S.
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container_title Acta crystallographica. Section D, Biological crystallography.
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creator García-Nafría, Javier
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description Homotrimeric dUTPases contain three active sites, each formed by five conserved sequence motifs originating from all three subunits. The essential fifth motif lies in a flexible C‐terminal arm which becomes ordered during catalysis and is disordered in most crystal structures. Previously, it has been shown that the two Bacillus subtilis dUTPases, YncF and YosS, differ from their orthologues in the position in the sequence of the essential Phe‐lid residue, which stacks against the uracil base, and in the conformation of the general base aspartate, which points away from the active site. Here, three structures of the complex of YncF with dU–PPi–Mg2+ and the structure of YosS complexed with dUMP are reported. dU–PPi–Mg2+ triggers the ordering of both the C‐terminal arm and a loop (residues 18–26) which is uniquely disordered in the Bacillus dUTPases. The dUMP complex suggests two stages in substrate release. Limited proteolysis experiments allowed those complexes in which C‐terminal cleavage is hindered and those in which it can be assumed to be ordered to be identified. The results lead to the suggestion that dUpNHpp is not a perfect substrate mimic, at least for the B. subtilis enzymes, and provide new insights into the mechanism of these two dUTPases in comparison to their orthologues. The enzyme mechanism is reviewed using the present and previous crystal structures as snapshots along the reaction coordinate.
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subjects Aspartates
Bacillus
Bacillus subtilis
Bacillus subtilis - enzymology
Bacteriology
Cleavage
Crystal structure
Crystallography, X-Ray
Deoxyuracil Nucleotides - chemistry
Deoxyuracil Nucleotides - metabolism
dU-PPi-Mg2+ complex
dUTPases
enzyme mechanism
Enzymes
Magnesium - chemistry
Magnesium - metabolism
Protein Conformation
Protein Folding
Pyrophosphatases - chemistry
Pyrophosphatases - metabolism
Residues
Stacks
transition-state pseudo-mimic
Uracil
title Tying down the arm in Bacillus dUTPase: structure and mechanism
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