The intrachain disulfide bridge is responsible of the unusual stability properties of novel acylphosphatase from Escherichia coli

Abstract

Acylphosphatase (AcP) activity in prokaryotes was classically attributed to some aspecific acid phosphatases. We identified an open reading frame for a putative AcP in the b0968 Escherichia coli gene and purified the recombinant enzyme after checking by RT-PCR that it was indeed expressed. EcoAcP has a predicted typical fold of the AcP family but displays a very low specific activity and a high structural stability differently from its mesophilic and similarly to its hyperthermophilic counterparts. Site directed mutagenesis suggests that, together with other structural features, the intrachain S-S bridge in EcoAcP is involved in a remarkable thermal and chemical stabilization of the protein without affecting its catalytic activity.

The Folding Process of Acylphosphatase from Escherichia coli is Remarkably Accelerated by the Presence of a Disulfide Bond

“…These data are in agreement with previously published results describing a mutational variant of EcoAcP in which only one of the two cysteine residues is substituted by an alanine residue. 26 Except for a few cases, 39 it has been generally reported that disulfide bonds are responsible for a stabilization of the overall three-dimensional structure of proteins of different sizes, regardless of their structural organization. [40][41][42][43][44][45][46][47] It is clear that the native disulfide bond of EcoAcP also has a marked influence on the folding process of this protein.…”

“…Such data are in agreement with previously reported results in which single cysteine EcoAcP variants were studied. 26 Folding and unfolding kinetics as a function of GdnHCl concentration…”

“…Nonionic chaotropic agents, such as urea, are not effective at any concentration. 26 Figure 3 shows the GdnHCl-induced unfolding curves of EcoAcP and mutEcoAcP measured at equilibrium at pH 5.5 and 28°C. Unfolding was monitored using both tryptophan intrinsic fluorescence and the CD signal at 228 nm.…”

“…26 This indicates that the presence of a disulfide bond is not a rare feature in this structural family, particularly in bacteria.…”

“…[19][20][21][22][23][24][25] A new AcP from Escherichia coli (EcoAcP) was recently isolated. 26 It is a small protein composed of 95 residues whose three-dimensional solution structure has been determined by NMR. 27 EcoAcP displays an α/β structure with a topology similar to that found in other members of the family so far studied (Fig.…”