Figure 5.
In vitro complementation of the fully reconstituted DEBS assembly line harboring an AT1-null mutation with the trans-acting KirCII AT in the presence of equimolar ethylmalonate and methylmalonate concentrations: (A) Schematic of the overall enzyme system and the expected product, 12-desmethyl-12-ethyl-6-dEB; (B) LC-MS analysis of product distribution ratios at varying KirCII concentrations. Because DEBS incorporates ethylmalonate naturally,12 EIC quantification of the 12-desmethyl-12-ethyl-6-dEB analogue also includes other ethyl-6-dEB analogues. For details regarding the identification of products and EIC quantification, see the Experimental Procedures. IS, internal standard, N-Boc-glutamic acid benzyl ester (11.6 μM).