TAILIEUCHUNG - Báo cáo khoa học: Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase

The initial step of tetrapyrrole biosynthesis inEscherichia coliinvolves 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 speci-ficityin vitro using a range of substrates and enzyme variants. | ỊFEBS Journal Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase Corinna Luer1 Stefan Schauer2 Simone Virus1 Wolf-Dieter Schubert3 Dirk W. Heinz3 Jurgen Moser1 and Dieter Jahn1 1 Institute of Microbiology TechnicalUniversity Braunschweig Germany 2 Institute of Molecular Biology and Biophysics Swiss FederalInstitute of Technology Zurich Switzerland 3 Division of StructuralBiology Helmholtz Centre for Infection Research Braunschweig Germany Keywords Escherichia coli glutamyl-tRNA glutamyl-tRNA reductase substrate recognition tetrapyrrole biosynthesis Correspondence D. Jahn Institute of Microbiology TechnicalUniversity Braunschweig Spielmannstrasse 7 D-38106 Braunschweig Germany Fax 49 531 391 5854 Tel 49 531 391 5801 E-mail Received 9 May 2007 revised 12 July 2007 accepted 13 July 2007 doi The 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 tRNAGlu 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 tRNAGlu by leucine and lysine respectively however did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNAGlu 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 .

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