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Sortase :Characterization, substrate specificity and mechanism.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Sortase :
其他題名:	Characterization, substrate specificity and mechanism.
作者:	Kruger, Ryan G.
面頁冊數:	157 p.
附註:	Source: Dissertation Abstracts International, Volume: 64-10, Section: B, page: 4919.
附註:	Supervisor: Dewey G. McCafferty.
Contained By:	Dissertation Abstracts International64-10B.
標題:	Chemistry, Biochemistry.
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3109196
ISBN:	049656739X

Sortase :Characterization, substrate specificity and mechanism.
Kruger, Ryan G.

Sortase :Characterization, substrate specificity and mechanism. [electronic resource] - 157 p.

Source: Dissertation Abstracts International, Volume: 64-10, Section: B, page: 4919.

Thesis (Ph.D.)--University of Pennsylvania, 2003.

Gram-positive bacteria display surface proteins on their cells walls to perform a variety of virulence related functions, including host surface attachment and immune system evasion. Many of these surface proteins are covalently linked to the peptidoglycan layer through the action of the sortase transpeptidases. The sortase enzymes recognize a conserved LPXTG motif in proteins targeted for cell wall anchoring, cleave the proteins between the threonine-glycine bond, and catalyze the transpeptidation to a peptidoglycan crosslinking spacer. While several studies have indicated that the sortase enzymes are critical for the establishment of infection in Gram-positive bacteria, there is little information about the kinetic mechanism or substrate specificity for this important family of enzymes.

ISBN: 049656739XSubjects--Topical Terms:

226900
Chemistry, Biochemistry.

Sortase :Characterization, substrate specificity and mechanism.
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520 # $a Gram-positive bacteria display surface proteins on their cells walls to perform a variety of virulence related functions, including host surface attachment and immune system evasion. Many of these surface proteins are covalently linked to the peptidoglycan layer through the action of the sortase transpeptidases. The sortase enzymes recognize a conserved LPXTG motif in proteins targeted for cell wall anchoring, cleave the proteins between the threonine-glycine bond, and catalyze the transpeptidation to a peptidoglycan crosslinking spacer. While several studies have indicated that the sortase enzymes are critical for the establishment of infection in Gram-positive bacteria, there is little information about the kinetic mechanism or substrate specificity for this important family of enzymes.
520 # $a Preliminary kinetic characterization of SrtA from S. aureus using an internally quenched fluorescence based assay has resulted in the underestimation of the true kinetic parameters for sortase. We have developed both FRET and HPLC based sortase activity assays that have allowed us to determine the true kinetic parameters, elucidate the kinetic mechanism and analyze the substrate specificity of SrtA.
520 # $a Previous reports have proposed that sortase utilizes a mechanism similar to the cysteine proteases. We show through a combination of steady-state, presteady-state, and FTIR MS/MS analysis that S. aureus SrtA proceeds through an acyl-enzyme intermediate using a kinetic mechanism consistent with a Ping-Pong hydrolytic shunt model. Our studies suggest that sortase proceeds through a mechanism similar to picornain 3C, in which an active site histidine residue activates a cysteine nucleophile by general base catalysis.
520 # $a There are two sortase isoforms in S. aureus, SrtA and SrtB. We have shown that SrtA is responsible for the display virulence factors containing the LPXTG recognition motif, while SrtB anchors surface proteins containing an NPQTN motif. Cell-based adhesion assays and protein A immunofluorescence assays have shown that SrtA anchors fibronectin binding protein and protein A respectively, and electron microscopy has shown that srtA - mutants have drastically altered cell wall morphologies while srtB- mutants appeared normal. We have also shown that SrtA is highly specific for the LPXTG recognition motif, using a 90-membered peptide substrate library.
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