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Báo cáo khoa học: Cloning and characterization of AtNUDT13, a novel mitochondrial Arabidopsis thaliana Nudix hydrolase specific for long-chain diadenosine polyphosphates

A cDNA corresponding to theAt3g26690 3 gene, which encodes a Nudix protein (AtNUDT13) with predicted mitochondrial localization, was iso-lated from an Arabidopsis thalianalibrary. The 202 amino acid AtNUDT13 polypeptide was overexpressed inEscherichia coliand purified to homo-geneity. | ỊFEBS Journal Cloning and characterization of AtNUDT13 a novel mitochondrial Arabidopsis thaliana Nudix hydrolase specific for long-chain diadenosine polyphosphates Kamil Olejnik1 Monika W. Murcha2 James Whelan2 and Elzbieta Kraszewska1 1 The Department of Plant Biochemistry Institute of Biochemistry and Biophysics Polish Academy of Sciences Warsaw Poland 2 Australian Research CouncilCentre of Excellence in Plant Energy Biology M316 The University of Western Australia Crawley Australia Keywords Arabidopsis thaliana diadenosine polyphosphates Nudix hydrolases diadenosine-5 5 -P1 P6-hexaphosphate diadenosine-5 5 -P1 P5-pentaphosphate Correspondence E. Kraszewska The Department of Plant Biochemistry Institute of Biochemistry and Biophysics Polish Academy of Sciences Pawinskiego 5A 02-106 Warsaw Poland Fax 48 22 6584804 Tel 48 22 5921218 E-mail elzbietak@ibb.waw.pl Received 28 June 2007 revised 18 July 2007 accepted 24 July 2007 doi 10.1111 j.1742-4658.2007.06009.x A cDNA corresponding to the At3g26690 gene which encodes a Nudix protein AtNUDT13 with predicted mitochondrial localization was isolated from an Arabidopsis thaliana library. The 202 amino acid AtNUDT13 polypeptide was overexpressed in Escherichia coli and purified to homogeneity. The preferred substrate for this hydrolase was diadenosine hexaphosphate Ap6A with Km and kcat Km values of 0.61 mM and 16.0 X 103 M 1-s-1 respectively. Optimal activity was at alkaline pH 8.5 with Mg2 5 mM as the cofactor. MS analysis revealed that the products of diadenosine hexaphosphate hydrolysis were ADP and adenosine tetraphosphate. Diadenosine pentaphosphate and adenosine tetraphosphate were additional substrates but diadenosine tetraphosphate and diadenosine triphosphate adenosine nucleotides diphosphoinositol polyphosphate and phosphoribosyl pyrophosphate were not hydrolyzed. Chemical crosslinking and size exclusion chromatography demonstrated that the protein exists as a monomer in solution. Subcellular localization .