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47
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Altering the sequence specificity of HaeIII methyltransferase by directed evolution using in vitro compartmentalization
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HIV protease was subjected to directed evolution, using both positive selection based on β-galactosidase cleavage, and negative selection based on the cytotoxicity of broad-specificity ('generalist') variants. The outcome was more specialized variants, rather than generalized as seen in most laboratory directed evolution experiments.
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James L.C., and Tawfik D.S. The specificity of cross-reactivity: promiscuous antibody binding involves specific hydrogen bonds rather than nonspecific hydrophobic stickiness. Protein Sci 12 (2003) 2183-2193
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James L.C., and Tawfik D.S. Structure and kinetics of a transient antibody binding intermediate reveal a kinetic discrimination mechanism in antigen recognition. Proc Natl Acad Sci USA 102 (2005) 12730-12735
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••]), the increase in OSBS activity, and the dramatic decrease in original AEE activity, were due to changes in active site residues that determine substrate specificity.
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••]), the increase in OSBS activity, and the dramatic decrease in original AEE activity, were due to changes in active site residues that determine substrate specificity.
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Several mutant aspartate aminotransferases (AATases) were characterized in order to evolve or design AATase with tyrosine aminotransferase (TATase) activity. The A293D mutation was found to be specificity determining, and was used to produce by rational design variants with narrow substrate specificity.
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Chow M.A., McElroy K.E., Corbett K.D., Berger J.M., and Kirsch J.F. Narrowing substrate specificity in a directly evolved enzyme: the A293D mutant of aspartate aminotransferase. Biochemistry 43 (2004) 12780-12787. Several mutant aspartate aminotransferases (AATases) were characterized in order to evolve or design AATase with tyrosine aminotransferase (TATase) activity. The A293D mutation was found to be specificity determining, and was used to produce by rational design variants with narrow substrate specificity.
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Seebeck F.P., and Hilvert D. Conversion of a PLP-dependent racemase into an aldolase by a single active site mutation. J Am Chem Soc 125 (2003) 10158-10159
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The article describes the introduction of a new activity (hydroxymandelate synthetase) into a homologous dioxygenase that shares the same substrate, and the first catalytic step (hydroxyphenylpyruvate). Interestingly, some of the designed libraries (saturation mutagenesis of selected active site residues) yielded a higher number of variants exhibiting the new activity, than those retaining the original one. Although the newly evolved variants show rather weak hydroxymandelate synthetase activity (≪1000-fold lower than the wild type enzyme), their specificity is completely reverted.
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O'Hare H.M., Huang F., Holding A., Choroba O.W., and Spencer J.B. Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution. FEBS Lett 580 (2006) 3445-3450. The article describes the introduction of a new activity (hydroxymandelate synthetase) into a homologous dioxygenase that shares the same substrate, and the first catalytic step (hydroxyphenylpyruvate). Interestingly, some of the designed libraries (saturation mutagenesis of selected active site residues) yielded a higher number of variants exhibiting the new activity, than those retaining the original one. Although the newly evolved variants show rather weak hydroxymandelate synthetase activity (≪1000-fold lower than the wild type enzyme), their specificity is completely reverted.
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Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase
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Hsu C.C., Hong Z., Wada M., Franke D., and Wong C.H. Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase. Proc Natl Acad Sci USA 102 (2005) 9122-9126
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Aharoni A., Gaidukov L., Yagur S., Toker L., Silman I., and Tawfik D.S. Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization. Proc Natl Acad Sci USA 101 (2004) 482-487
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Draganov D.I., Teiber J.F., Speelman A., Osawa Y., Sunahara R., and La Du B.N. Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities. J Lipid Res 46 (2005) 1239-1247
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Miller B.G., and Raines R.T. Identifying latent enzyme activities: substrate ambiguity within modern bacterial sugar kinases. Biochemistry 43 (2004) 6387-6392
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Several bacterial sugar kinases with ambiguous substrate specificity were identified by a genetic complementation screen. These kinases, possessing a weak glucokinase activity, were recruited by a glucokinase-deficient E. coli strain, and provided it with increased metabolic capacity. Higher metabolic efficiency evolved through a mutation in the promoter region. The article therefore illustrates the fact that changes in regulation comprise a key step in the divergence of a new function.
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Miller B.G., and Raines R.T. Reconstitution of a defunct glycolytic pathway via recruitment of ambiguous sugar kinases. Biochemistry 44 (2005) 10776-10783. Several bacterial sugar kinases with ambiguous substrate specificity were identified by a genetic complementation screen. These kinases, possessing a weak glucokinase activity, were recruited by a glucokinase-deficient E. coli strain, and provided it with increased metabolic capacity. Higher metabolic efficiency evolved through a mutation in the promoter region. The article therefore illustrates the fact that changes in regulation comprise a key step in the divergence of a new function.
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Promiscuous activities of three members of amidohydrolase superfamily - dihydroorotase (DHO), phosphotriesterase (PTE), and PTE-homology protein (PHP) - were examined, and a partially overlapping pattern of promiscuity was found. Directed evolution has been applied to increase the esterase activity of PTE and PHP. The results reveal structural and evolutionary relationships within these superfamily members.
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Roodveldt C., and Tawfik D.S. Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily. Biochemistry 44 (2005) 12728-12736. Promiscuous activities of three members of amidohydrolase superfamily - dihydroorotase (DHO), phosphotriesterase (PTE), and PTE-homology protein (PHP) - were examined, and a partially overlapping pattern of promiscuity was found. Directed evolution has been applied to increase the esterase activity of PTE and PHP. The results reveal structural and evolutionary relationships within these superfamily members.
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Hoffmeister D., Yang J., Liu L., and Thorson J.S. Creation of the first anomeric D/L-sugar kinase by means of directed evolution. Proc Natl Acad Sci USA 100 (2003) 13184-13189
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Timar E., Groma G., Kiss A., and Venetianer P. Changing the recognition specificity of a DNA-methyltransferase by in vitro evolution. Nucleic Acids Res 32 (2004) 3898-3903
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Evolution of enzymatic activities in the orotidine 5′-monophosphate decarboxylase suprafamily: enhancing the promiscuous D-arabino-hex-3-ulose 6-phosphate synthase reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase
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Yew W.S., Akana J., Wise E.L., Rayment I., and Gerlt J.A. Evolution of enzymatic activities in the orotidine 5′-monophosphate decarboxylase suprafamily: enhancing the promiscuous D-arabino-hex-3-ulose 6-phosphate synthase reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase. Biochemistry 44 (2005) 1807-1815
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