The long term goals of the present application are to precisely determine the identities of all the proteins and small molecules in pathogenic bacteria that are enzymatically acetylated and to similarly define the identities of all the proteins in yeast that are myristoylated. We propose to do this using our recently developed activity-based protein profiling method. In the case of the "acetylome", we will use chloroacetyl-CoA, a reagent that we discovered and showed that it can be used as a substrate by every Gcn5-related N-acetyltransferase (GNAT's) that we have tested to date. By enzymatically transferring the chloroacetyl group to the protein, this chemically "marks" that protein as a substrate for acetylation, and that mark can be chemically reacted with thiol-containing compounds to generate stable covalent species that can be purified and identified. While our earlier studies were performed with pure or relatively pure mixtures of proteins, in order to confirm that neither specificity nor regioselectivity were compromised with the reagent, we have since demonstrated that the method can be used in crude extracts of cells with equal efficiency and selectivity. The reagent is equally useful in the preparation of bisubstrate analogues that exhibit nanomolar binding affinity to the corresponding GNAT. We will thus establish all of the substrates for all the GNATs in selected organisms. Having identified all the substrates, we will then prepare bisubstrate analogues containing these substrates coupled to solid supports and use them as "bait" to identify the cognate GNAT. Those GNATs that are essential for cellular viability will be subjected to detailed enzymatic and structural analysis. In the case of the "myristoylome", we have shown that undecynoyl-CoA is an efficient substrate for the Saccharomyces cerevisiae protein - N-myristoyl-transferase, a member of the GNAT superfamily. This substrate will cause the undecynoylation of the 1-N-terminal glycine of what we estimate is 60 protein substrates, based on amino terminal sequence in yeast. Using azide-containing fluorophores and affinity purification tags, we will identify which of these 60 proteins is, in fact, myristoylated. In both projects, we will use two- dimensional SDS-PAGE/IEF to separate the labeled protein mixtures, and the identification of the proteins will be made using trypsin hydrolysis followed by HPLC-MS/MS. We have significant experience in these methods, having recently used them to define the many potential targets for isoniazid in Mycobacterium tuberculosis crude cell extracts.
The GNAT superfamily is one of the largest protein superfamilies and is responsible for acetylation, succinylation and myristoylation of both small molecules and proteins. A large number of these are implicated in regulation and in human disease.
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|Serrano, Hector; Blanchard, John S (2013) Kinetic and isotopic characterization of L-proline dehydrogenase from Mycobacterium tuberculosis. Biochemistry 52:5009-15|
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|Vergnolle, Olivia; Xu, Hua; Blanchard, John S (2013) Mechanism and regulation of mycobactin fatty acyl-AMP ligase FadD33. J Biol Chem 288:28116-25|
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|Wolfson-Stofko, Brett; Hadi, Timin; Blanchard, John S (2013) Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis. Arch Biochem Biophys 540:53-61|
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|Sikora, Alison L; Wilson, Daniel J; Aldrich, Courtney C et al. (2010) Kinetic and inhibition studies of dihydroxybenzoate-AMP ligase from Escherichia coli. Biochemistry 49:3648-57|
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