The Aes Protein and the Monomeric α-Galactosidase fromEscherichia coli Form a Non-covalent Complex

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 ␣-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 ␣-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.The Escherichia coli aes (or ybac) gene encodes the 36-kDa cytoplasmic protein Aes (1), a carboxylesterase belonging to the hormone-sensitive lipase family (HSL) 1 (2). Although the physiological role of this enzyme is still obscure, it has recently received greater attention due to the discovery that Aes is involved in the regulation of the maltose regulon in E. coli (reviewed in Ref. 3). The E. coli maltose system consists of 10 genes encoding proteins for the uptake and metabolism of maltodextrin and maltose (3). MalT is the transcriptional activator of the maltose regulon and the prototype of a new family of transcription factors (4, 5) and integrates several signals through its amino-terminal three domains (6). To activate mal genes transcription MalT acts together with the cyclic AMPcatabolite protein complex, the inducer maltotriose, and ATP (7). Several regulatory circuits modulate the activity of MalT, e.g. MalT expression is regulated by Mlc, a glucose-inducible repressor (8). In addition, MalT is negatively controlled by at least three proteins: MalY, MalK, and Aes. MalY is an enzyme with cystathionine ␤-lyase activity (9, 10), which ...

Biochemical and Thermostability Features of Acetyl Esterase Aes from Escherichia coli

“…In a previous report [5] we confirmed the kinetic parameters of the acetyl-esterase Aes, obtained from Kanaya et al [2], at 25 °C and using pNP-butanoate as substrate. Because a complete biochemical characterization of Aes has never been reported we seek to fill this gap.…”

“…In fact it has been demonstrated that Aes negatively controls the activity of MalT, the transcriptional activator of the maltose regulon, by a direct protein-protein interaction [4]. Our group recently suggested a similar potential role of Aes in the regulation of the raffinose regulon via a specific protein-protein interaction with -galactosidase [5]. However, the physiological role of Aes in E. coli is still unknown; insertion mutations in aes gene do not result in any easily discernable phenotype and it has been reported that the basal level of Aes expression is very low [1].…”

“…Finally, cells were harvested by centrifugation (6,000xg, 4 °C, 10 min), washed with 25 mM Tris-HCl buffer (pH 8.5)/ 2.5 mM MgCl 2 / 0.5 mM EDTA and stored at -20 °C. Purification of the recombinant enzymes were performed as previously reported [5]. Briefly, after cellular disruption with a French Pressure Cell (Aminco, USA), the crude extract was treated with (NH 4 ) 2 SO 4 to obtain the selective precipitation of Aes.…”

Interaction between β-lactoglobulin and lactase and its effect on enzymatic activity

“…On the other hand, Mandrich, Caputo, Martin, Rossi, and Manco (2002) reported that the activity of the E. coli Aes protein, a carboxylesterase, increases six-fold when it binds to a 50 kDa a-galactosidase produced by the same bacterium. In this case the activating effect is due to a protein-protein interaction.…”

Use of surface‐enhanced laser desorption/ionization ‐time of flight to explore bacterial proteomes

“…These papers mainly describe the use of the ProteinChip Arrays with purified proteins [10][11][12][13][14], or extracted surface proteins [15][16][17][18][19]. A few reports are available in the literature in which this technology is used with protein extracts [20][21][22].…”