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Structural and biochemical analyses of a Clostridium perfringens sortase D transpeptidase.

Suryadinata R, Seabrook SA, Adams TE, Nuttall SD, Peat TS - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues.Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif.Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, 343 Royal Parade, Parkville, Victoria 3052, Australia.

ABSTRACT
The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99 Å resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents.

No MeSH data available.


Related in: MedlinePlus

CpSrtD recognizes and cleaves the LPQTGS sorting motif in vitro. (a) Recombinant CpSrtD was incubated in the absence (lane 1) or presence of Aβ1–16 peptides fused to LPETG (lane 2), LPNTGS (lane 3), LPQTGS (lane 4) or LAETG (lane 5) sorting motifs. (b) Catalytic efficiency of recombinant CpSrtD towards Aβ1–16-LPQTGS substrate was measured at room temperature (RT, lane 2), 303 K (lane 3), 310 K (lane 4), 316 K (lane 5) or 323 K (lane 6). (c) CpSrtD was pre-incubated in the absence (lane 1) or presence of EDTA at a concentration of 1 mM (lane 2), 2 mM (lane 3), 5 mM (lane 4), 10 mM (lane 5), 20 mM (lane 6), 50 mM (lane 7), 100 mM (lane 8) or 200 mM (lane 9). EDTA-treated CpSrtD was then incubated with Aβ1–16-LPQTGS and the ability to form the thioacyl intermediate was measured. (d) EDTA-treated (100 mM) CpSrtD was incubated with Aβ1–16-LPQTGS in the absence of metal ions (lane 1) or the presence of 10 mM Ca2+ (lane 2), Cu2+ (lane 3), Co2+ (lane 4), Fe3+ (lane 5), Mg2+ (lane 6), Mn2+ (lane 7), Ni2+ (lane 8) or Zn2+ (lane 9). The formation of thioacyl intermediate in (a), (b) and (c) was analysed by Western blot using mouse α-­Aβ (WO2) antibody (top panels), and equal loading was assessed using mouse α-His5 antibody (bottom panels).
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fig3: CpSrtD recognizes and cleaves the LPQTGS sorting motif in vitro. (a) Recombinant CpSrtD was incubated in the absence (lane 1) or presence of Aβ1–16 peptides fused to LPETG (lane 2), LPNTGS (lane 3), LPQTGS (lane 4) or LAETG (lane 5) sorting motifs. (b) Catalytic efficiency of recombinant CpSrtD towards Aβ1–16-LPQTGS substrate was measured at room temperature (RT, lane 2), 303 K (lane 3), 310 K (lane 4), 316 K (lane 5) or 323 K (lane 6). (c) CpSrtD was pre-incubated in the absence (lane 1) or presence of EDTA at a concentration of 1 mM (lane 2), 2 mM (lane 3), 5 mM (lane 4), 10 mM (lane 5), 20 mM (lane 6), 50 mM (lane 7), 100 mM (lane 8) or 200 mM (lane 9). EDTA-treated CpSrtD was then incubated with Aβ1–16-LPQTGS and the ability to form the thioacyl intermediate was measured. (d) EDTA-treated (100 mM) CpSrtD was incubated with Aβ1–16-LPQTGS in the absence of metal ions (lane 1) or the presence of 10 mM Ca2+ (lane 2), Cu2+ (lane 3), Co2+ (lane 4), Fe3+ (lane 5), Mg2+ (lane 6), Mn2+ (lane 7), Ni2+ (lane 8) or Zn2+ (lane 9). The formation of thioacyl intermediate in (a), (b) and (c) was analysed by Western blot using mouse α-­Aβ (WO2) antibody (top panels), and equal loading was assessed using mouse α-His5 antibody (bottom panels).

Mentions: One notable feature that is observed in most sortases is their ability to preferentially recognize a specific signal motif for catalysis. To identify the signal motif preferred by C. perfringens sortase to achieve efficient catalysis, we performed a series of in vitro transpeptidation reactions using a substrate that consists of the first 16 amino-acid residues of the amyloid-β (Aβ1–16) peptide fused at the C-terminus with the LPETG, LPNTGS, LPQTGS or LAETG sorting motifs. This panel of substrates represents signal motifs that are recognized by the different classes of sortase family, including class A (LPETG), class D (LPNTGS and LPQTGS) and class E (LAETG). Western blot analyses using anti-Aβ antibody to detect the CpSrtD–substrate thioacyl intermediate revealed no CpSrtD catalytic activity towards the class E signal motif (Fig. 3 ▸a, lane 5, top panel) and minimal activity towards either the LPETG (Fig. 3 ▸a, lane 2, top panel) or LPNTGS (Fig. 3 ▸a, lane 3, top panel) signal motifs. In contrast, recombinant CpSrtD showed a strong preference towards the LPQTGS motif (Fig. 3 ▸a, lane 4, top panel).


Structural and biochemical analyses of a Clostridium perfringens sortase D transpeptidase.

Suryadinata R, Seabrook SA, Adams TE, Nuttall SD, Peat TS - Acta Crystallogr. D Biol. Crystallogr. (2015)

CpSrtD recognizes and cleaves the LPQTGS sorting motif in vitro. (a) Recombinant CpSrtD was incubated in the absence (lane 1) or presence of Aβ1–16 peptides fused to LPETG (lane 2), LPNTGS (lane 3), LPQTGS (lane 4) or LAETG (lane 5) sorting motifs. (b) Catalytic efficiency of recombinant CpSrtD towards Aβ1–16-LPQTGS substrate was measured at room temperature (RT, lane 2), 303 K (lane 3), 310 K (lane 4), 316 K (lane 5) or 323 K (lane 6). (c) CpSrtD was pre-incubated in the absence (lane 1) or presence of EDTA at a concentration of 1 mM (lane 2), 2 mM (lane 3), 5 mM (lane 4), 10 mM (lane 5), 20 mM (lane 6), 50 mM (lane 7), 100 mM (lane 8) or 200 mM (lane 9). EDTA-treated CpSrtD was then incubated with Aβ1–16-LPQTGS and the ability to form the thioacyl intermediate was measured. (d) EDTA-treated (100 mM) CpSrtD was incubated with Aβ1–16-LPQTGS in the absence of metal ions (lane 1) or the presence of 10 mM Ca2+ (lane 2), Cu2+ (lane 3), Co2+ (lane 4), Fe3+ (lane 5), Mg2+ (lane 6), Mn2+ (lane 7), Ni2+ (lane 8) or Zn2+ (lane 9). The formation of thioacyl intermediate in (a), (b) and (c) was analysed by Western blot using mouse α-­Aβ (WO2) antibody (top panels), and equal loading was assessed using mouse α-His5 antibody (bottom panels).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4498605&req=5

fig3: CpSrtD recognizes and cleaves the LPQTGS sorting motif in vitro. (a) Recombinant CpSrtD was incubated in the absence (lane 1) or presence of Aβ1–16 peptides fused to LPETG (lane 2), LPNTGS (lane 3), LPQTGS (lane 4) or LAETG (lane 5) sorting motifs. (b) Catalytic efficiency of recombinant CpSrtD towards Aβ1–16-LPQTGS substrate was measured at room temperature (RT, lane 2), 303 K (lane 3), 310 K (lane 4), 316 K (lane 5) or 323 K (lane 6). (c) CpSrtD was pre-incubated in the absence (lane 1) or presence of EDTA at a concentration of 1 mM (lane 2), 2 mM (lane 3), 5 mM (lane 4), 10 mM (lane 5), 20 mM (lane 6), 50 mM (lane 7), 100 mM (lane 8) or 200 mM (lane 9). EDTA-treated CpSrtD was then incubated with Aβ1–16-LPQTGS and the ability to form the thioacyl intermediate was measured. (d) EDTA-treated (100 mM) CpSrtD was incubated with Aβ1–16-LPQTGS in the absence of metal ions (lane 1) or the presence of 10 mM Ca2+ (lane 2), Cu2+ (lane 3), Co2+ (lane 4), Fe3+ (lane 5), Mg2+ (lane 6), Mn2+ (lane 7), Ni2+ (lane 8) or Zn2+ (lane 9). The formation of thioacyl intermediate in (a), (b) and (c) was analysed by Western blot using mouse α-­Aβ (WO2) antibody (top panels), and equal loading was assessed using mouse α-His5 antibody (bottom panels).
Mentions: One notable feature that is observed in most sortases is their ability to preferentially recognize a specific signal motif for catalysis. To identify the signal motif preferred by C. perfringens sortase to achieve efficient catalysis, we performed a series of in vitro transpeptidation reactions using a substrate that consists of the first 16 amino-acid residues of the amyloid-β (Aβ1–16) peptide fused at the C-terminus with the LPETG, LPNTGS, LPQTGS or LAETG sorting motifs. This panel of substrates represents signal motifs that are recognized by the different classes of sortase family, including class A (LPETG), class D (LPNTGS and LPQTGS) and class E (LAETG). Western blot analyses using anti-Aβ antibody to detect the CpSrtD–substrate thioacyl intermediate revealed no CpSrtD catalytic activity towards the class E signal motif (Fig. 3 ▸a, lane 5, top panel) and minimal activity towards either the LPETG (Fig. 3 ▸a, lane 2, top panel) or LPNTGS (Fig. 3 ▸a, lane 3, top panel) signal motifs. In contrast, recombinant CpSrtD showed a strong preference towards the LPQTGS motif (Fig. 3 ▸a, lane 4, top panel).

Bottom Line: Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues.Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif.Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, 343 Royal Parade, Parkville, Victoria 3052, Australia.

ABSTRACT
The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99 Å resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents.

No MeSH data available.


Related in: MedlinePlus