TAILIEUCHUNG - Báo cáo khoa học: Expression, purification and catalytic activity of Lupinus luteus asparagine b-amidohydrolase and its Escherichia coli homolog

We describe the expression, purification, and biochemical characterization of two homologous enzymes, with amido-hydrolase activities, of plant (Lupinus luteuspotassium-independent asparaginase, LlA) and bacterial (Escherichia coli, ybiK/spt/iaaA gene product, EcAIII) origin. Both enzymes were expressed inE. colicells, with (LlA) or with-out (EcAIII) aHis-tag sequence. The proteins were purified, yielding6 or 30 mgÆL )1 of culture, respectively. | Eur. J. Biochem. 271 3215-3226 2004 FEBS 2004 doi Expression purification and catalytic activity of Lupinus luteus asparagine p-amidohydrolase and its Escherichia coli homolog Dominika Borek1 Karolina Michalska1 Krzysztof Brzezinski1 Agnieszka Kisiel2 Jan Podkowinski2 David T. Bonthron3 Daniel Krowarsch4 Jacek Otlewski4 and Mariusz Jaskolski1 2 1 Department of Crystallography Faculty of Chemistry A. Mickiewicz University Poznan Poland 2Center for Biocrystallographic Research Institute of Bioorganic Chemistry Polish Academy of Sciences Poznan Poland 3Molecular Medicine Unit University of Leeds UK 4Laboratory of Protein Engineering Institute of Biochemistry and Molecular Biology Wroclaw University Poland We describe the expression purification and biochemical characterization of two homologous enzymes with amidohydrolase activities of plant Lupinus luteus potassium-independent asparaginase LlA and bacterial Escherichia coli ybiK spt iaaA gene product EcAIII origin. Both enzymes were expressed in E. coli cells with LlA or without EcAIII a His-tag sequence. The proteins were purified yielding 6 or 30 mg L-1 of culture respectively. The enzymes are heat-stable up to 60 C and show both isoaspartyl dipeptidase and L-asparaginase activities. Kinetic parameters for both enzymatic reactions have been determined showing that the isoaspartyl peptidase activity is the dominating one. Despite sequence similarity to aspartylglucosaminidases no aspartylglucosaminidase activity could be detected. Phylogenetic analysis demonstrated the relationship of these proteins to other asparaginases and aspartylglucosaminidases and suggested their classification as N-terminal nucleophile hydrolases. This is consistent with the observed autocatalytic breakdown of the immature proteins into two subunits with liberation of an N-terminal threonine as a potential catalytic residue. Keywords asparaginase isoaspartyl peptidase aspartylglu-cosaminidase Ntn-hydrolase .

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