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dc.contributor.authorLüer, Corinna
dc.contributor.authorSchauer, Stefan
dc.contributor.authorVirus, Simone
dc.contributor.authorSchubert, Wolf-Dieter
dc.contributor.authorHeinz, Dirk W
dc.contributor.authorMoser, Jürgen
dc.contributor.authorJahn, Dieter
dc.date.accessioned2008-05-26T10:42:47Z
dc.date.available2008-05-26T10:42:47Z
dc.date.issued2007-09
dc.identifier.citationGlutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase. 2007, 274 (17):4609-14 FEBS J.en
dc.identifier.issn1742-464X
dc.identifier.pmid17697121
dc.identifier.doi10.1111/j.1742-4658.2007.05989.x
dc.identifier.urihttp://hdl.handle.net/10033/28052
dc.description.abstractThe initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.
dc.language.isoenen
dc.subject.meshAldehyde Oxidoreductasesen
dc.subject.meshBase Sequenceen
dc.subject.meshCatalysisen
dc.subject.meshChromatography, High Pressure Liquiden
dc.subject.meshDNA Primersen
dc.subject.meshEscherichia colien
dc.subject.meshGlutamic Aciden
dc.subject.meshHydrogenen
dc.subject.meshKineticsen
dc.subject.meshMutagenesis, Site-Directeden
dc.titleGlutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase.en
dc.typeArticleen
dc.contributor.departmentInstitute of Microbiology, Technical University Braunschweig, Germany.en
dc.identifier.journalThe FEBS journalen
refterms.dateFOA2008-09-05T00:00:00Z
html.description.abstractThe initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.


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