In vitro biosynthesis of molecular warheads from hydroxamate- and β-lactone-containing protease inhibitors
Biochemistry
Microbial Ecology and Applied Microbiology
Final Report Abstract
Proteases have been established as drug targets for various diseases e.g. myeloma, diabetes, hypertension and viral infections. Notably, the development of protease inhibitor drug candidates has greatly been inspired by the discovery of natural products. They often exhibit their inhibitory properties by binding to target enzymes with specific warheads e.g. electrophilic epoxyketones, aldehydes, β-lactones or to inhibit metalloproteases metal-chelating hydroxamate moieties. We have recently identified the biosynthetic pathways of the N-hydroxy-alkylsuccinamic acid-containing metalloprotease inhibitors actinonin and matylstatins as well as the 2-carboxy-3-alkyl β-lactone containing proteasome inhibitors cystargolides and belactosins. In the DFG funded research project we proposed to study the formation of these warheads in detail using in vitro biochemistry and protein crystallography. While the investigation of the N-hydroxy-alkyl-succinamic acid warhead proved to be unsuccessful through various attempts, substantial progress was made in understanding the biosynthesis of the β-lactone moiety of cystargolides and belactosins. We found that three enzymes are involved in the assembly of this warhead. The SAM-dependent methyltransferase CysG methylates 3-isopropylmalate (3-IPM) to generate the cryptic 3-IPM-1-methyl ester (Met-3-IPM). This intermediate is cyclized in an ATP-dependent manner to the β-lactone methyl ester (CysF) and further processed by the methylesterase CysE to generate the β-lactone carboxylic acid building block. We propose that CysG acts as a diverter, committing the primary metabolite 3-IPM to cystargolide biosynthesis and masking the 1-carboxylic acid group for the following lactonization. In total we determined only five specific enzymes to be essential for cystargolide assembly: the SAM-dependent methyltransferase CysG, the methylesterase CysE, the ATP-grasp enzyme CysD and the adenylating enzymes CysC and CysF. In belactosin biosynthesis we find the same mechanism for the formation of the β-lactone warhead. Here, it is the CysG homolog BelI that generates a cryptic 1-methylester intermediate from 3-sec butylmalate. Again an adenylating enzyme and a methylesterase are involved in subsequent reactions. Next, we characterized BelI and CysG by crystallography, computational analysis, mutagenesis, and activity assays. Thereby, we identified a His-His-Asp (HHD) motif in the active sites of the two enzymes, which is crucial for binding a catalytically active calcium ion. This conserved divalent metal-dependent mechanism distinguishes BelI and CysG from previously characterized O-methyltransferases.
Publications
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Structure of Staphylococcus aureus ClpP Bound to the Covalent Active‐Site Inhibitor Cystargolide A. Angewandte Chemie International Edition, 63(3).
Illigmann, Astrid; Vielberg, Marie‐Theres; Lakemeyer, Markus; Wolf, Felix; Dema, Taulant; Stange, Patrik; Kuttenlochner, Wolfgang; Liebhart, Elisa; Kulik, Andreas; Staudt, Nicole D.; Malik, Imran; Grond, Stephanie; Sieber, Stephan A.; Kaysser, Leonard; Groll, Michael & Brötz‐Oesterhelt, Heike
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Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG. Journal of Biological Chemistry, 300(1), 105507.
Beller, Patrick; Fink, Phillipp; Wolf, Felix; Männle, Daniel; Helmle, Irina; Kuttenlochner, Wolfgang; Unterfrauner, Daniel; Engelbrecht, Alicia; Staudt, Nicole D.; Kulik, Andreas; Groll, Michael; Gross, Harald & Kaysser, Leonard
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Deciphering the SAM- and metal-dependent mechanism of O-methyltransferases in cystargolide and belactosin biosynthesis: A structure–activity relationship study. Journal of Biological Chemistry, 300(9), 107646.
Kuttenlochner, Wolfgang; Beller, Patrick; Kaysser, Leonard & Groll, Michael
