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Dissertation/Thesis Abstract

Molecular recognition and catalysis in sortase A from Staphylococcus aureus
by Bentley, Matthew Lawrence, Ph.D., University of Pennsylvania, 2008, 258; 3328693
Abstract (Summary)

Gram-positive bacteria utilize surface displayed proteins to mediate a wide range of virulence-associated functions. Inhibition of surface protein display has thus emerged as a potential target for the development of novel anti-infective therapeutics. Sortase transpeptidases catalyze the covalent attachment of many of these surface proteins to the Gram-positive cell wall. In Staphylococcus aureus, the Sortase A (SrtA) transpeptidase recognizes putative surface proteins that contain a C-terminal LPXTG sorting motif. SrtA specifically cleaves this sequence between the threonine and glycine residues, then catalyzes formation of an amide bond between the threonine and the pentaglycine crossbridge of branched lipid II. A second sortase, SrtB, catalyzes a similar reaction but anchors a substrate containing an NPQTN motif. Although recent studies have shed light on the kinetic and catalytic mechanisms of SrtA, the molecular basis for substrate specificity is poorly understood. Similarly, attempts to discover potent SrtA inhibitors with in vivo efficacy have to date met with limited success.

To explore the molecular basis for substrate recognition in SrtA and SrtB, we used domain swapping to engineer NPQTN specificity into SrtA. Based on our results, we identified the β6/β7 loop region of SrtA as an important region for determining specificity. Subsequent mutagenesis localized the recognition domain in SrtA to hydrophobic residues Val168 and Leu169, which interact with the Leu-Pro sequence of the substrate.

The role of a conserved active site arginine residue (Arg197) in SrtA catalysis has been a source of much debate. To better understand its role in catalysis, we used native chemical ligation to replace Arg197 with citrulline, a non-ionizable analog. Kinetic studies with this mutant indicate that Arg197 utilizes a hydrogen bond to promote catalysis, rather than an electrostatic interaction as previously thought.

Based on our findings, we propose an expanded model for LPXTG recognition by SrtA that involves both hydrophobic interactions between Val168/Leu169 and the Leu-Pro sequence of the sorting signal as well as a hydrogen bond interaction with Arg197. These interactions combine to stabilize LPXTG binding and promote catalysis. This information will guide future efforts to design potent SrtA inhibitors as novel anti-infective therapeutics.

Indexing (document details)
Advisor: McCafferty, Dewey G.
School: University of Pennsylvania
School Location: United States -- Pennsylvania
Source: DAI-B 69/09, Dissertation Abstracts International
Subjects: Molecular biology, Biochemistry, Biophysics
Keywords: Arginine reductase, Molecular recognition, Sortase, Substrate specificity
Publication Number: 3328693
ISBN: 978-0-549-81131-2
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