Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys 184 , His 185 , and Asp 213 catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His 85 , His 122 , and Glu 90 metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8 -12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDSpolyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.Every organism responds to a sudden increase in the environmental temperature by the overexpression of a set of highly conserved heat shock proteins (1, 2). Most of these heat shock proteins function either as molecular chaperones, assisting in protein folding and renaturation or as proteases which degrade proteins that are beyond rescue.Hsp31, 1 the hchA gene product (formerly known as YedU), is a heat-inducible homodimeric protein of 31-kDa subunits, which was recently shown to exhibit molecular chaperone activity (3, 4). It promotes the functional folding of citrate synthase, ␣-glucosidase, and alcohol dehydrogenase. It also prevents the aggregation at 43°C of citrate synthase and alcohol dehydrogenase and interacts specifically with unfolded proteins (3, 4). Although Hsp31 does not exhibit any ATPase activity, some of its chaperone activities are partially inhibited by ATP (3, 4). The crystal structure of Hsp31 was solved at 1.6 Å resolution and revealed a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded protein substrates and expla...
EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. EstU1 assumes a β-lactamase-like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β-lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β-lactam antibiotics. This result explains the weak β-lactamase activity of EstU1 compared with class C β-lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum.
Poren zeigen Größe: Ein mesoporöses metall‐organisches Netzwerk, das vorrangig aus Tb3+‐Ionen und tripodalen Carboxylatliganden aufgebaut ist, bringt es auf Käfige mit 3.9 und 4.7 nm Durchmesser (siehe Bild). Das leere Netzwerk ist beständig und kann Gas‐ oder Ferrocenmoleküle aufnehmen, was durch Gassorptions‐ und Lumineszenzstudien bestätigt wurde.
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