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PMID:12374803

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Citation

Mandrich, L, Caputo, E, Martin, BM, Rossi, M and Manco, G (2002) The Aes protein and the monomeric alpha-galactosidase from Escherichia coli form a non-covalent complex. Implications for the regulation of carbohydrate metabolism. J. Biol. Chem. 277:48241-7

Abstract

Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the alpha-galactosidase from E. coli. The E. coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because alpha-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli.

Links

PubMed Online version:10.1074/jbc.M207398200

Keywords

Acetylesterase; Amino Acid Sequence; Base Sequence; Carbohydrate Metabolism; Cloning, Molecular; DNA Primers; Escherichia coli/enzymology; Escherichia coli/metabolism; Escherichia coli Proteins; Kinetics; Molecular Sequence Data; Protein Binding; Proteins/chemistry; Proteins/genetics; Proteins/isolation & purification; Proteins/metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; alpha-Galactosidase/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:AES

acts_upstream_of_or_within

GO:0051346: negative regulation of hydrolase activity

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

ECOLI:AES

involved_in

GO:0051346: negative regulation of hydrolase activity

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

See also

References

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