Andrew C. Mercer

Fluorescent Profiling of Modular Biosynthetic Enzymes by Complementary Metabolic and Activity Based Probes

The study of the enzymes responsible for natural product biosynthesis has proven a valuable source of new enzymatic activities and been applied to a number of biotechnology applications. Protein profiling could prove highly complementary to genetics based approaches by allowing us to understand the activity, transcriptional control, and post-translational modification of these enzymes in their native and dynamic proteomic environments. Here we present a method for the fluorescent profiling of PKS, NRPS, and FAS multidomain modular synthases in their whole proteomes using complementary metabolic and activity based probes. After first examining the reactivity of these activity based probes with a variety of purified recombinant PKS, NRPS, and FAS enzymes in vitro, we apply this duel labeling strategy to the analysis of modular synthases in a human breast cancer cell line and two strains of the natural product producer Bacillus subtilis. Collectively, these studies demonstrate that complementary protein profiling approaches can prove highly useful in the identification and assignment of inhibitor specificity and domain structure of these modular biosynthetic enzymes.

In vivo modification of native carrier protein domains.

Insider information: Selective labeling of endogenous proteins within cells has been an elusive goal. Here carrier protein labeling has been optimized for visualization, isolation, and protein sequencing.

Antibiotic evaluation and in vivo analysis of alkynyl Coenzyme A antimetabolites in Escherichia coli.

Pantothenamides have been the subject of much study as potential inhibitors of CoA and carrier protein dependent biosynthetic pathways. Based on an initial observation of growth inhibition in Escherichia coli by 3, we have synthesized a small panel of pantetheine analogues and re-examined the inhibitory properties of this class of antibiotics with an emphasis on understanding the ability of these compounds to act as substrates of native CoA and carrier protein utilizing biosynthetic pathways. Our findings suggest that a secondary structure-activity relationship is an important factor in the antibiotic activity of these compounds.

Fluorescent multiplex analysis of carrier protein post-translational modification.

Multiplex analysis has proven to be a powerful tool for dissecting genetic identities within complex biological mixtures, as illustrated by applications in in situ hybridization, polymorphic PCR, and gene-expression profiling. Given the progression of proteomic analyses, comparable systems will be a valuable asset for protein-based applications. An ideal system for the application of multiplex analysis to proteins is the promiscuous post-translational modification of carrier-protein domains from thioester-mediated biosyntheses. This transformation, catalyzed by 4’-phosphopantetheinyltransferases (PPTases), can be modified with a variety of reporter-labeled prosthetic groups to yield labeled proteins, wherein each chemical species transfers with a unique kinetic profile. Here we introduce a three-color fluorescent multiplex analysis of carrier-protein domains that may be applied to the discovery of primary and secondary biosynthetic pathways and as functional markers for fusion-protein systems involving multiple species. Carrier-protein domains comprise a small yet diverse group of proteins essential to primary and secondary biosynthetic pathways, including fatty acid, polyketide, and non-ribosomal peptide synthesis. The carrier proteins involved in these pathways function as scaffolds to mediate modular synthesis through a domain bearing a 4’-phosphopantetheine arm. This prosthetic group is incorporated through transfer of 4’phosphopantetheine from coenzyme A (CoA) to a conserved serine residue by a PPTase (Figure 1). The three-component reaction between CoA, carrier protein, and PPTase has been shown to accept a variety of CoA–thioester substrates. Recently, we further investigated this tolerance by covalently modifying carrier-protein domains with reporter molecules, as illustrated by the conversion of apo-carrier protein to cryptocarrier protein (Figure 1). This modification has been subsequently utilized in fusion-protein labeling with in vivo applications.