Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Escherichia coli strain WP2uvrA is widely used in general mutagenicity screening tests because of its high sensitivity to many kinds of mutagens and it serves as a supplement to the standard Salmonella typhimurium tester strains. In contrast to Salmonella His(+) revertants, E.coli Trp(+) revertants have not been characterized at the molecular level. In this study we found that in the trpE65 allele of WP2uvrA the triplet that codes for the fourth amino acid from the N-terminus of anthranilate synthetase was an ochre stop codon (TAA) instead of a glutamine codon (CAA). In spontaneous Trp(+) revertants the ochre codon had been changed to glutamine (CAA), lysine (AAA), glutamic acid (GAA), leucine (TTA), serine (TCA) or tyrosine (TAC, TAT). Since tryptophan prototrophy could also be restored by ochre suppressor mutations at the anticodon sites in the genes for tRNA(Glu) (glnU), tRNA(Lys) (lysT) and tRNA(Tyr) (tyrT, tyrU), the Trp(+) reversion system with E.coli WP2uvrA detected five types of base substitutions, A.T-->T.A, A.T-->C.G, A.T-->G.C, G.C-->A.T and G.C-->T.A. About 30-50% of Trp(+) revertants induced by N-ethyl-N'-nitro-N-nitrosoguanidine, captan and angelicin plus UVA irradiation were attributable to reversion at the trpE65 ochre locus; the others were attributable to suppressor mutations. In contrast, almost all revertants induced by N-methyl-N'-nitro-N-nitrosoguanidine, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and furylfuramide were caused by suppressor mutations. Thus, the high mutagen sensitivity of WP2uvrA is due to several target sites consisting of A.T base pairs (trpE65, lysT) and G.C base pairs (glnU, tyrT, tyrU).
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PMID:Characterization of Trp(+) reversions in Escherichia coli strain WP2uvrA. 1211 Jun 27

Aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase) are Escherichia coli paralogs that share 43% sequence identity. A plausible model posits that TATase arose from a duplication of an ancestral AATase-like enzyme. Directed evolution of AATase to an enzyme having TATase activity was undertaken in order to compare the evolved AATase variants with homologous TATases. Eight rounds of DNA shuffling and in vivo selection followed by a backcross with WT AATase produced enzymes that exhibited 100-270-fold increases in k(cat)/K(m)(Phe) and had as much as 11% of the tyrosine aminotransferase activity of WT E.coli TATase. Amino acid substitutions in 11 clones from rounds 7 and 8 were compared with conserved residues in AATases and TATases. The findings are conveniently and compactly illustrated by the use of Venn diagrams and set theory notation. A statistically significant (0.001<or=p<or=0.008) concentration of mutations occurs in a subset of positions (set AAT-TAT) that is conserved (>or=75% identical) in AATases and variable (<75% identical) in TATases. Very few mutations occur in the intersection (set AAT intersection TAT) of amino acid residues that are conserved in both enzyme types. Seven mutations from set AAT-TAT were combined by site-directed mutagenesis to give a construct that is 60% as active as the best round 8 enzyme, which has 13 amino acid replacements. The Venn diagrams may provide a generally useful tool to highlight the most important specificity determinants for rational redesign. Amino acid replacements were mapped onto the crystal structure of a hydrocinnamate complex of a designed TATase. Five of the seven positions most frequently substituted in the evolved clones are within 15 A of the phenyl side-chain, but only six of the 48 positions that were mutated once or twice are within that radius. Context dependence, neutral mutations, different selective pressures, and stochastic components provide explanations for the observation that many of the substitutions found in the directly evolved enzymes differ from the corresponding amino acids found in the modern natural TATases.
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PMID:How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase. 1263 55