Single Mutation on the Surface of Staphylococcus aureus Sortase A Can Disrupt Its Dimerization

Abstract

Staphylococcus aureus Sortase A (SrtA) is an important Gram-positive membrane enzyme which catalyzes the anchoring of many cell surface proteins conserved with the LPXTG sequence. Recently SrtA has been demonstrated to be a dimer with a Kd of 55 microM in vitro. Herein, we show that a single point mutation of amino acid residue on the surface of SrtA can completely disrupt the dimerization. Native polyacrylamide gel electrophoresis and analytical gel filtration chromatography were used to detect the dimer-monomer equilibrium of SrtA mutants. Circular dichroism spectrum experiments were performed to study the conformational change of each SrtA mutant. An enzyme activity assay confirmed that all the SrtA mutants were active in vitro. Our results not only are important for understanding the SrtA protein self-associating mechanism but also provided the necessary starting materials for the study of sortase A pathway in vivo, which may have significant implications for discovering microbial physiology and give a potential target for novel Gram-positive antibiotics.

Site‐specific Protein Cross‐Linking with Genetically Incorporated 3,4‐Dihydroxy‐L‐Phenylalanine

“…The lack of copious cross-linking at residue K206 indicates the absence of nucleophiles that are in the close proximity to this residue and available to react with l -DOPA moiety. The yields of cross-linked dimer from K137DOPA (Figure 3, lane 3) and K206DOPA (Figure 3, lane 6) are consistent with the results from the point-mutation studies[12] (Figures S1 and S2). The data presented here are the first direct evidence to confirm the physical interaction of SrtA at the residue level.…”

“…Successful cross-linking supports our recent discovery of SrtA ΔN59 dimerization. [10,12] Furthermore, we have shown that the efficiency of the cross-linking does not cost its selectivity. The information from serial cross-linking experiments at different residues using the same method would allow us to fairly accurately map the dimerization domain of SrtA.…”

“…A point mutation on its surface, K137A, has been shown to disrupt the dimerization of SrtA ΔN59 without a significant change in protein conformation. [12] Thus, it is highly likely that this residue is involved in dimerization of SrtA ΔN59 by establishing a selective electrostatic interaction with the residue(s) on the surface of the binding SrtA ΔN59 counterpart. The cognate residue is a potential nucleophile that can attack the ortho -quinone intermediate formed by the oxidation of l -DOPA.…”

One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst

“…S3, line 4). The formation of SrtA dimer is resulted from a disulfide bridge of two active site Cys184 residues located in enzyme under denaturing conditions without adding reducing reagent 55 . The eluate was dialyzed and lyophilized to yield 407.2 μg of protein, which suggested that 91% of the SrtA was immobilized onto the IDA-nickel magnetic particles according to the amount of SrtA in 5 mL of supernatant (449.0 μg).…”

In-vitro evaluation of the anti-bacterial properties of crude extracts of Dorstenia mannii (Moraceae)

“…With new diseases and increasing resistance to existing drugs, there is need to discover and develop new innovating drugs with diminished side effects to combat bacteria infections (Cirzrt et al, 2005;Zhu et al, 2008). The use of herbs to treat diseases is almost universal among non industrialised societies (Brater and Daly, 2000).…”