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PMID:15265041

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Contents

Citation

Borek, D, Michalska, K, Brzezinski, K, Kisiel, A, Podkowinski, J, Bonthron, DT, Krowarsch, D, Otlewski, J and Jaskolski, M (2004) Expression, purification and catalytic activity of Lupinus luteus asparagine beta-amidohydrolase and its Escherichia coli homolog. Eur. J. Biochem. 271:3215-26

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.

Links

PubMed Online version:10.1111/j.1432-1033.2004.04254.x

Keywords

Amino Acid Sequence; Aspartylglucosylaminase/chemistry; Aspartylglucosylaminase/genetics; Aspartylglucosylaminase/isolation & purification; Aspartylglucosylaminase/metabolism; Catalysis; Enzyme Stability; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/isolation & purification; Escherichia coli Proteins/metabolism; Kinetics; Lupinus/enzymology; Lupinus/genetics; Molecular Sequence Data; Molecular Structure; Phylogeny; Protein Denaturation; Sequence Alignment; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity; Temperature

Significance

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Useful Materials and Methods

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Annotations

Gene product Qualifier GO ID GO term name Evidence Code with/from Aspect Notes Status

iaaA:Gene_Product(s)

Deprecated

GO:0006530

asparagine catabolic process

IDA: Inferred from Direct Assay

P

IaaA can hydrolyze asparagine to aspartate + NH3 in vitro, but the affinity for asparagine is lower than that of known bacterial asparaginases, so this activity may not be physiologically relevant.[1]

complete

iaaA:Gene_Product(s)

GO:0004067

asparaginase activity

IDA: Inferred from Direct Assay

F

See notes for the annotation of IaaA toGO:0006530.

complete

iaaA:Gene_Product(s)

GO:0016787

hydrolase activity

IDA: Inferred from Direct Assay

F

complete

iaaA:Gene_Product(s)

Deprecated

GO:0009063

cellular amino acid catabolic process

IDA: Inferred from Direct Assay

P

See notes for the annotation of IaaA toGO:0006530.

complete

iaaA:Gene_Product(s)

GO:0008798

beta-aspartyl-peptidase activity

IDA: Inferred from Direct Assay

F

Table 2

complete

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See also

References

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  1. Michalska, K et al. (2005) Crystal structure of isoaspartyl aminopeptidase in complex with L-aspartate. J. Biol. Chem. 280 28484-91 PubMed EcoliWiki page