Skip to main content
construction release_alert
Scholars@Duke will be undergoing maintenance April 11-15. Some features may be unavailable during this time.
cancel

Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus.

Publication ,  Journal Article
Bentley, ML; Lamb, EC; McCafferty, DG
Published in: The Journal of biological chemistry
May 2008

The Staphylococcus aureus transpeptidase sortase A (SrtA) is responsible for anchoring a range of virulence- and colonization-associated proteins to the cell wall. SrtA recognizes substrates that contain a C-terminal LPXTG motif. This sequence is cleaved following the threonine, and an amide bond is formed between the threonine and the pentaglycine cross-bridge of branched lipid II. Previous studies have implicated the beta6/beta7 loop region of SrtA in LPXTG recognition but have not systematically characterized this domain. To better understand the individual roles of the residues within this loop, we performed alanine-scanning mutagenesis. Val-168 and Leu-169 were found to be important for substrate recognition, and Glu-171 was also found to be important, consistent with its hypothesized role as a Ca(2+)-binding residue. Gly-167 and Asp-170 were dispensable for catalysis, as was Gln-172. The role of Arg-197 in SrtA has been the subject of much debate. To explore its role in catalysis, we used native chemical ligation to generate semi-synthetic SrtA in which we replaced Arg-197 with citrulline, a non-ionizable analog. This change resulted in a decrease of <3-fold in k(cat)/K(m), indicating that Arg-197 utilizes a hydrogen bond, rather than an electrostatic interaction. Our results are consistent with a model for LPXTG recognition wherein the Leu-Pro sequence is recognized primarily by hydrophobic contacts with SrtA Val-168 and Leu-169, as well as a hydrogen bond from Arg-197. This model contradicts the previously proposed mechanism of binding predicted by the x-ray crystal structure of SrtA.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

The Journal of biological chemistry

DOI

EISSN

1083-351X

ISSN

0021-9258

Publication Date

May 2008

Volume

283

Issue

21

Start / End Page

14762 / 14771

Related Subject Headings

  • Temperature
  • Substrate Specificity
  • Staphylococcus aureus
  • Protein Structure, Tertiary
  • Protein Denaturation
  • Peptidyl Transferases
  • Mutation
  • Mutagenesis
  • Models, Molecular
  • Kinetics
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Bentley, M. L., Lamb, E. C., & McCafferty, D. G. (2008). Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus. The Journal of Biological Chemistry, 283(21), 14762–14771. https://doi.org/10.1074/jbc.m800974200
Bentley, Matthew L., Erin C. Lamb, and Dewey G. McCafferty. “Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus.The Journal of Biological Chemistry 283, no. 21 (May 2008): 14762–71. https://doi.org/10.1074/jbc.m800974200.
Bentley ML, Lamb EC, McCafferty DG. Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus. The Journal of biological chemistry. 2008 May;283(21):14762–71.
Bentley, Matthew L., et al. “Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus.The Journal of Biological Chemistry, vol. 283, no. 21, May 2008, pp. 14762–71. Epmc, doi:10.1074/jbc.m800974200.
Bentley ML, Lamb EC, McCafferty DG. Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus. The Journal of biological chemistry. 2008 May;283(21):14762–14771.

Published In

The Journal of biological chemistry

DOI

EISSN

1083-351X

ISSN

0021-9258

Publication Date

May 2008

Volume

283

Issue

21

Start / End Page

14762 / 14771

Related Subject Headings

  • Temperature
  • Substrate Specificity
  • Staphylococcus aureus
  • Protein Structure, Tertiary
  • Protein Denaturation
  • Peptidyl Transferases
  • Mutation
  • Mutagenesis
  • Models, Molecular
  • Kinetics