Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs.

The discovery of peptide substrates for enzymes with exclusive, selective activities is a central goal in chemical biology. In this paper, we develop a hybrid computational and biochemical method to rapidly optimize peptides for specific, orthogonal biochemical functions. The method is an iterative machine learning process by which experimental data is deposited into a mathematical algorithm that selects potential peptide substrates to be tested experimentally. Once tested, the algorithm uses the experimental data to refine future selections. This process is repeated until a suitable set of de novo peptide substrates are discovered. We employed this technology to discover orthogonal peptide substrates for 4’-phosphopantetheinyl transferase, an enzyme class that covalently modifies proteins. In this manner, we have demonstrated that machine learning can be leveraged to guide peptide optimization for specific biochemical functions not immediately accessible by biological screening techniques, such as phage display and random mutagenesis.

Ketosynthases (KSs) catalyze carbon–carbon bond-forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping-pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester-tethered substrates to the active sites of KSs. Therefore, protein–protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here, we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate-engaged AcpP–FabF complexes, one of which provides the missing AcpP–FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine-scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism, which advances future engineering efforts in both FASs and evolutionarily related PKSs.

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Nonribosomal peptide synthetases (NRPSs) are responsible for the synthesis of a variety of bioactive natural products with clinical and economic significance. Interestingly, these large multimodular enzyme machineries incorporate nonproteinogenic d-amino acids through the use of auxiliary epimerization domains, converting l-amino acids into d-amino acids that impart into the resulting natural products unique bioactivity and resistance to proteases. Due to the large and complex nature of NRPSs, several questions remain unanswered about the mechanism of the catalytic domain reactions. We have investigated the use of mechanism-based crosslinkers to probe the mechanism of an epimerization domain in gramicidin S biosynthesis. In addition, MD simulations were performed, showcasing the possible roles of catalytic residues within the epimerization domain.