TAILIEUCHUNG - Báo cáo khoa học: High resolution structure and catalysis of O-acetylserine sulfhydrylase isozyme B from Escherichia coli

The crystal structure of the dimericO-acetylserine sulfhydrylase isozyme B from Escherichia coli(CysM), complexed with the substrate analog citrate, has been determined at A˚ resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O-acetylserine, whereas the C6-carboxylate adopted two conformations. | ễFEBS Journal High resolution structure and catalysis of O-acetylserine sulfhydrylase isozyme B from Escherichia coli Georg Zocher Ulrich Wiesand and Georg E. Schulz Institut fur Organische Chemie und Biochemie Albert-Ludwigs-Universitat Freiburg im Breisgau Germany Keywords biosynthesis of L-cysteine enzymatic assay homodimer asymmetry nonstandard L-amino acids X-ray diffraction Correspondence G. E. Schulz Institut fur Organische Chemie und Biochemie Albert-Ludwigs-Universitat Albertstr. 21 79104 Freiburg im Breisgau Germany Fax 49 761 203 6161 Tel 49 761 203 6058 E-mail Website http . Received 24 July 2007 revised 22 August 2007 accepted 23 August 2007 doi The crystal structure of the dimeric O-acetylserine sulfhydrylase isozyme B from Escherichia coli CysM complexed with the substrate analog citrate has been determined at A resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O-acetylserine whereas the C6-carboxylate adopted two conformations. The activity of the enzyme and of several active center mutants was determined using an assay based on O-acetylserine and thio-nitrobenzoate TNB . The unnatural substrate TNB was modeled into the reported structure. The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spectrum of the enzyme. A comparison of the reported structure with previously published CysM structures revealed large conformational changes. One of the crystal forms contained two dimers each of which comprised one subunit in a closed and one in an open conformation. Although the homodimer asymmetry was most probably caused by crystal packing it indicates that the enzyme can adopt such a state in solution which may be relevant for the catalytic reaction. In bacteria archaea and plants the biosynthesis of .

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