Figure 4
The BioV protein cleaves the ester group of pimeloyl-ACP methyl ester.
(a) Schematic diagram of the enzymatic reaction catalysed by H. pylori BioV. (b) Enzymatic assays for H. pylori BioV hydrolysis of pimeloyl-ACP methyl ester to pimeloyl-ACP. The enzymatic reactions (10 μl total volume) were performed at 37 °C and contained 100 μM pimeloyl-ACP methyl ester as substrate. The product is the slower-migrating pimeloyl-ACP, which can be resolved from the substrate9,17 in a destabilizing urea-PAGE system (Materials and Methods). The minus signs denote reactions containing all components except BioV. The triangle over the right-hand six lanes of the bottom panel represents BioV concentrations in an inverse dilution series (0.3, 1, 3, 10, 30 and 100 nM). (c) Substrate specificity of BioV. The minus sign denotes no addition of BioV whereas the plus sign denotes addition of the enzyme. The triangle represents an inverse BioV dilution series (3,10, 30 and 100 nM). (d) Mass spectrometry of H. pylori holo-ACP. Two forms of the protein are found due to oxidization of a methionine reside to the sulphoxide during purification. Form 1 (mass 8990.0) is the native form whereas form 2 (mass 9005.8) is the oxidized form. (e) Mass spectrometry of pimeloyl-ACP methyl ester (Me-pimeloyl-ACP). Form 1 (mass 9146.6) is the native form whereas form 2 (mass 9162.6) is the oxidized form. (f) Mass spectrum of the reaction products of panel B shows cleavage of pimeloyl-ACP methyl ester (Me-pimeloyl-ACP) (9,146.2 or 9162.6 amu) to pimeloyl-ACP (mass 9,132.5 or 9148.2 amu).
