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Query: EC:3.1.3.5 (5'-nucleotidase)
 3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DNA sequence of the ushA gene, encoding UDP-sugar hydrolase (5'-nucleotidase), has been determined. The amino-terminal sequence encodes a signal peptide whose predicted processing site is confirmed by N-terminal amino acid analysis of purified mature UshA protein. The signal sequence contains a concentration of rare codons in comparison with the mature sequence. The origins of transcription from the ushA promoter have been determined, using primer extension. Three transcripts, originating within a 6 bp region, were identified and might be related to three overlapping potential -10 hexamers in the ushA promoter region. There was a discernable change in the relative proportion of these transcripts during growth-phase regulation of the ushA gene.
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PMID:Nucleotide sequence and transcriptional analysis of the E. coli ushA gene, encoding periplasmic UDP-sugar hydrolase (5'-nucleotidase): regulation of the ushA gene, and the signal sequence of its encoded protein product. 301 67

We show that Escherichia coli produce a factor that inhibits the activity of tyrosine and serine/threonine protein kinases. The factor is a protein found in the periplasmic compartment and is also secreted into the culture medium. Using a particle concentration fluorescence immunoassay specific for tyrosine kinase activity and inhibition of the tyrosine kinase p56(lck), we purified this factor to apparent homogeneity. Analysis of trypsin-digested fragments by mass spectrometry identified the inhibitor as the bacterial periplasmic protein UDP-sugar hydrolase, an enzyme with potent and nonspecific 5'-nucleotidase activity. Overexpression of the enzyme in bacteria leads to coordinate increases in both 5'-nucleotidase and p56(lck) inhibitory activity, confirming the identity of the inhibitor. The kinase inhibitory activity appears to be due to the formation of adenosine, which we show is inhibitory for p56(lck), cAMP-dependent protein kinase, and casein kinase. Overexpression of UDP-sugar hydrolase leads to an increase in the recovery of enteropathogenic E. coli following infection of HeLa cell monolayers and corresponding alterations in tyrosine-phosphorylated host proteins. These results suggest that UDP-sugar hydrolase may be an important factor affecting host cell function following intracellular bacterial infection.
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PMID:Identification of a bacterial inhibitor of protein kinases. Mechanism and role in host cell invasion. 879 49

The crystal structure of 5'-nucleotidase (5'-NT) from E. coli, also known as UDP-sugar hydrolase, has been determined at 1.7 A resolution. Two zinc ions are present in the active site, which is located in a cleft between two domains. The dimetal center and a catalytic Asp-His dyad are the main players in the catalytic mechanism. Structure-based sequence comparisons show that the structure also provides a model for animal 5'-NTs, which together with other ectonucleotidases terminate the action of nucleotides as extracellular signaling substances in the nervous system.
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PMID:X-ray structure of the Escherichia coli periplasmic 5'-nucleotidase containing a dimetal catalytic site. 1033 72

Two mutational mechanisms, both supported by experimental studies, have been proposed for the evolution of new or improved enzyme specificities in bacteria. One mechanism involves point mutation(s) in a gene conferring novel substrate specificity with partial or complete loss of the original (wild-type) activity of the encoded product. The second mechanism involves gene duplication followed by silencing (inactivation) of one of these duplicates. Some of these 'silent genes' may still be transcribed and translated but produce greatly reduced levels of functional protein; gene silencing, in this context, is distinct from the more common associations with bacterial partitioning sequences, and with genes which are no longer transcribed or translated. Whereas most Salmonella enterica strains are ushA(+), encoding an active 5'-nucleotidase (UDP-sugar hydrolase), some natural isolates, including most genetically related strains of serotype Typhimurium, have an ushA allele (designated ushA(c)) which produces a protein with, comparatively, very low 5'-nucleotidase activity. Previous sequence analysis of cloned ushA(c) and ushA(+) genes from serotype Typhimurium strain LT2 and Escherichia coli, respectively, did not reveal any changes which might account for the significantly different 5'-nucleotidase activities. The mechanism responsible for this reduced activity of UshA(c) has hitherto not been known. Sequence analysis of Salmonella ushA(+) and ushA(c) alleles indicated that the relative inactivity of UshA(c) may be due to one, or more, of four amino acid substitutions. One of these changes (S139Y) is in a sequence motif that is conserved in 5'-nucleotidases across a range of diverse prokaryotic and eukaryotic species. Site-directed mutagenesis confirmed that a Tyr substitution of Ser-139 in Salmonella UshA(+) was solely responsible for loss of 5'-nucleotidase activity. It is concluded that the corresponding single missense mutation is the cause of the UshA(c) phenotype. This is the first reported instance of gene inactivation in natural isolates of bacteria via a missense mutation. These results support a model of evolution of new enzymes involving a 'silent gene' which produces an inactive, or relatively inactive, product, and are also consistent with the evolution of a novel, but unknown, enzyme specificity by a single amino acid change.
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PMID:The cryptic ushA gene (ushA(c)) in natural isolates of Salmonella enterica (serotype Typhimurium) has been inactivated by a single missense mutation. 1142 65

E. coli UshA, a bifunctional enzyme with UDP-sugar hydrolase and 5'-nucleotidase activities, is secreted to the periplasm but has a specific protein inhibitor located in the cytoplasm. It has been previously suggested that some 5'-nucleotidase, or a folded domain of this enzyme, may be active in the cytoplasm prior to export. If true, the intracellular inhibitor may have a role in protecting the cell from the likely deleterious effects of any intracellular UshA activity. Using deletion mutagenesis to remove the UshA signal peptide, we have shown that the resulting UshA derivative is an active cytoplasmic 5'-nucleotidase, and causes conditional lethality. Our results support the hypothesis that the physiological role of the UshA inhibitor is to protect the intracellular nucleotide pool from any cytoplasmic 5'-nucleotidase activity.
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PMID:The role of the intracellular inhibitor of periplasmic UDP-sugar hydrolase (5'-nucleotidase) in Escherichia coli: cytoplasmic localisation of 5'-nucleotidase is conditionally lethal. 1180 43

Nucleoside 5'-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In Yersinia intermedia suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDP-alcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25 degrees C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5'-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product (UshA(Yi)) of the Y. intermedia ushA gene (ushA(Yi)) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of Escherichia coli (UshA(Ec)). Therefore, UshA(Ec) was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshA(Ec) and UshA(Yi) behaved similarly. Besides being the first study of a UshA enzyme in the genus Yersinia, this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with k(cat)/K(m) ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshA(Ec) conformers and the respective homology models constructed for UshA(Yi), and also in relation to possible biological functions. Interestingly, every Yersinia species with a sequenced genome contains an intact ushA gene, except Y. pestis, which in all its sequenced biovars contains a ushA gene inactivated by frameshift mutations.
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PMID:CDP-alcohol hydrolase, a very efficient activity of the 5'-nucleotidase/UDP-sugar hydrolase encoded by the ushA gene of Yersinia intermedia and Escherichia coli. 1864 Nov 43