A Bottom‐Up Proteomic Approach to Identify Substrate Specificity of Outer‐Membrane Protease OmpT. Issue 52 (10th October 2017)

• Record Type: Journal Article
• Authors: Wood, Sarah E.; Sinsinbar, Gaurav; Gudlur, Sushanth; Nallani, Madhavan; Huang, Che‐Fan; Liedberg, Bo; Mrksich, Milan
• Is Part Of: Angewandte Chemie international edition. Volume 56:Issue 52(2017)
• Page Start: 16531
• Page End: 16535
• Publication Date: 2017-10-10
• Subjects: chemical biology -- peptides -- protease activity -- proteomics -- SAMDI-MS; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.201707535
• Languages: English
• ISSNs: 1433-7851

Abstract

Abstract: Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer‐membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1′) and nearest‐neighbor positions (P2, P2′) and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400‐fold improvement in OmpT catalytic efficiency, with a k cat / K m value of 6.1×10 6 L mol −1 s −1 . Wild‐type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme. Abstract : OmpT substrate optimization : Substrate specificity of a bacterial outer‐membrane protease OmpT is determined through use of peptide arrays and SAMDI‐MS, a label‐free high‐throughput mass‐spectrometry‐based assay. By screening hundreds of peptide substrates, it is possible to identify an optimal peptide sequence that dramatically improves the catalytic efficiency of the protease. Prod=product, Subst=substrate.