title: Expression, purification and catalytic activity of Lupinus luteus asparagine beta-amidohydrolase and its Escherichia coli homolog.
abstract: 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 degrees 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.
authors.affiliation: Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland.
authors.forename: Dominika
authors.initials: D
authors.lastname: Borek
authors.forename: Karolina
authors.initials: K
authors.lastname: Michalska
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Brzezinski
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Kisiel
authors.forename: Jan
authors.initials: J
authors.lastname: Podkowinski
authors.forename: David T
authors.initials: DT
authors.lastname: Bonthron
authors.forename: Daniel
authors.initials: D
authors.lastname: Krowarsch
authors.forename: Jacek
authors.initials: J
authors.lastname: Otlewski
authors.forename: Mariusz
authors.initials: M
authors.lastname: Jaskolski
mesh_terms: D000595:Amino Acid Sequence; D001227:Aspartylglucosylaminase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D031297:Lupinus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010802:Phylogeny; D011489:Protein Denaturation; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D001227:Aspartylglucosylaminase