Characterization of the mutX gene of Streptococcus pneumoniae as a homologue of Escherichia coli mutT, and tentative definition of a catalytic domain of the dGTP pyrophosphohydroiases

Abstract

We show that deletion of a gene of Streptococcus pneumoniae, which we call mutX, confers a mutator phenotype to resistance to streptomycin. Analysis of the DNA sequence changes that occurred in several streptomycin-resistant mutants showed that mutations are unidirectional AT to CG transversions. The mutX gene is located immediately downstream of the previously identified ung gene and genetic evidence suggests that the two genes are co-ordinately regulated. Nucleotide sequence determination reveals that the mutX gene encodes a 17,870 Da protein (154 residues) which exhibits significant homology with the MutT protein of Escherichia coli, a nucleoside triphosphatase (dGTP pyrophosphohydrolase). The mutX gene complements the E. coli mutT mutator phenotype when introduced on a plasmid. Site-directed mutagenesis and analysis of nitrosoguanidine-induced mutT mutants suggest that a small region of high homology between the two proteins (61% identity over 23 residues) is part of the catalytic site of the nucleoside triphosphatase. Computer searching for sequence homology to MutX uncovered a second E. coli protein, the product of orf17, a gene of unknown function located near the ruvC gene. The region of high homology between MutX and MutT is also conserved in this protein, which raises the interesting possibility that the orf17 gene plays some role in determining mutation rates in E. coli. Finally, a small set of proteins, including a family of virus-encoded proteins and two evolutionarily conserved proteins encoded by an antisense transcript from the Xenopus laevis and human bFGF genes, were also found to harbour significant homology to this highly conserved region.

The hDcp2 protein is a mammalian mRNA decapping enzyme

“…A modified polyethyleneimine-cellulose TLC was used to develop the reaction products in 0.75 M LiCl. We found that this running buffer resolved m 7 GDP from 32 Pi, which otherwise comigrate in the standard 0.45 M (NH 4 ) 2 SO 4 buffer (18). As shown in Fig.…”

“…The Nudix motif was originally identified in a class of pyrophosphatase proteins and shown to be critical for pyrophosphatase activity (31,32). The Nudix motif consists of a highly conserved 23-aa sequence, GX 5 EX 7 REUXEEXGU, where X can be any amino acid and U is an aliphatic hydrophobic amino acid (31,32). To test whether the hDcp2 Nudix motif was functional, the two conserved glutamic acid residues (E147 and E148) within this motif were substituted by glutamine.…”

“…Decapping products were resolved by polyethyleneimine-cellulose TLC plates as described in Wang and Kiledjian (18) except the TLC was developed in 0.75 M LiCl. Cold standards and 32 Pi were loaded alongside the samples and visualized by UV shadowing or autoradiography, respectively. The nucleotide diphosphate kinase (NDPK) reactions were carried out as described (18).…”

Functional Significance of the Conserved Residues for the 23-Residue Module among MTH1 and MutT Family Proteins

“…Genes for MutT homolog proteins with dGTPase or 8-oxodGTPase activity were identified in Proteus vulgaris and Streptococcus pneumoniae, bacteria distantly related to E. coli (16,17). All three MutT homolog proteins have molecular masses ranging from 13 to 18 kDa, and the degree of identity ranges from 19 to 40%.…”

Cloning and Characterization of the First Member of the Nudix Family from Arabidopsis thaliana

“…This motif was first described as a MutT motif in a MutT protein from Escherichia coli (2). MutT is a nucleoside triphosphatase that hydrolyzes all canonical nucleoside triphosphates with a preference for deoxyguanosine triphosphate (dGTP) and its oxidized form 7,8-dihydro-8-oxodeoxyguanosine .…”