Co-opting CoA biosynthesis for the in vivo modification of carrier proteins

Polyketides and non-ribosomal peptides are a rich source of bioactive compounds. These natural products, as well as fatty acids, are made by carrier proteins dependant synthases. In these biosynthetic pathways the carrier protein functions to isolate building blocks from the cytoplasm and deliver growing intermediates to the proper enzyme active site. The carrier protein mediates this tethering via a posttranslationly attached 4'phosphopantetheine arm. This arm is derived from Co- enzyme A. Our goal was to manipulate the posttranslational modification of the carrier protein domain in order to modify, identify, and isolate carrier proteins and their synthases from natural product producing organisms. Initially we used CoA derivatives to modify carrier proteins in vitro. However, we have found that both in vitro and in vivo, pantetheine analogs can be converted into CoA analogs using the native CoA biosynthetic pathway. Using this knowledge we made a panel of pantetheine analogs and tested them for the ability to be processed by the CoA biosynthetic machinery as well as the ability to modify carrier proteins in vivo. We found that a wide range of pantetheine analogs can be converted into CoA analogs in vitro, but only analogs that have a good kinetic profile with the first enzyme in the CoA biosynthetic pathway, panK, are competitive enough with natural substrates to modify carrier proteins in vivo. From our studies of pantetheine analogs we were able to select analogs for in vivo labeling of carrier proteins in their native systems. A fluorescent pantetheine analog was used to modify the human fatty acid synthase in the SKBR3 cell line. Additionally, using a azide terminal pantetheine analog we were able to modify native carrier proteins from a number of bacteria. These in vivo modified carrier proteins can be visualized via gel or pulled-down via affinity label after reaction with an alkyne reporter. Mass spectral analysis of excised carrier proteins from SDS-PAGE allows for identification of the carrier proteins. This technique allowed us to identify and characterize the previously unreported acyl-carrier protein from Brevibacillus Brevi