TAILIEUCHUNG - Báo cáo khoa học: Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L)

cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5¢- and 3¢-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed inEscherichia coliwith a C-terminal penta His-tag. | ỊFEBS Journal Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop Pecten maximus L Andre Muller Frank JanBen and Manfred K. Grieshaber Institut fur Zoophysiologie Heinrich-Heine-Universitat Dusseldorf Germany Keywords catalytic triad octopine dehydrogenase ODH reaction mechanism site-directed mutagenesis Correspondence A. Muller Institut fur Zoophysiologie Heinrich-Heine-Universitat 40225 Dusseldorf Germany Fax 49 211 811 3897 Tel 49 211 811 5116 E-mail Database The nucleotide sequence reported has been submitted to the DDBJ EMBL GenBank database under the accession number AJ237916 Received 10 May 2007 revised 19 September 2007 accepted 17 October 2007 doi cDNA for octopine dehydrogenase ODH from the adductor muscle of the great scallop Pecten maximus was cloned using 5 - and 3 -RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH-5His was purified to homogeneity using metal-chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop s adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148 which is conserved in all opine dehydrogenases known to date was converted to serine or alanine showing that this residue is not intrinsically important for catalysis. His212 Arg324 and Asp329 which are also conserved in all known opine dehydrogenase sequences were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in Km and decreases in kcat

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