Back to Search
Start Over
Expression, purification and catalytic activity of Lupinus luteus asparagine β-amidohydrolase and its Escherichia coli homolog.
- Source :
- European Journal of Biochemistry; Aug2004, Vol. 271 Issue 15, p3215-3226, 12p
- Publication Year :
- 2004
-
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<superscript>−1</superscript> 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. [ABSTRACT FROM AUTHOR]
- Subjects :
- LUPINUS luteus
AMIDASES
ESCHERICHIA coli
ISOASPARTIC acid
GLYCOASPARAGINASE
PHYLOGENY
Subjects
Details
- Language :
- English
- ISSN :
- 00142956
- Volume :
- 271
- Issue :
- 15
- Database :
- Complementary Index
- Journal :
- European Journal of Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 13834590
- Full Text :
- https://doi.org/10.1111/j.1432-1033.2004.04254.x