Mechanism-based crosslinking as a gauge for functional interaction of modular synthases

Current Opinion in Structural Biology

2013

Naturally occurring polyketides and non-ribosomal peptides with broad and potent biological activities continue to inspire the discovery of new and improved analogs. The biosynthetic apparatus responsible for the construction of these natural products has been the target of intensive protein engineering efforts. Traditionally, engineering has focused on substituting individual enzymatic domains or entire modules with those of different building block specificity, or by deleting various enzymatic functions, in an attempt to generate analogs. This review highlights strategies based on site-directed mutagenesis of substrate binding pockets, semi-rational mutagenesis, and whole-gene random mutagenesis to engineer the substrate specificity, activity, and protein interactions of polyketide and non-ribosomal peptide biosynthetic machinery.

Section: Protein:protein Interactionsmentioning

confidence: 99%

Engineering polyketide synthases and nonribosomal peptide synthetases

Current Opinion in Structural Biology

2013

“…The difficulty of probing and interrogating the weak and transient interactions between ACP's and KS's is exemplified by the ongoing elegant work of Burkart and co-workers, which relies on the design and synthesis of specific mechanism-based probes that covalently cross-link the ACP and cognate interaction partner. 4,5,29,30 Clearly, additional tools need to be developed and evaluated for studying protein interactions among FAS's and PKS's, particularly for those biosynthetic systems with unusual substrate specificities, whereby effective cross-linkers might be difficult to discover. Macromolecular interactions of proteins in other biological systems have often been probed via the introduction of photocross-linking unnatural amino acids.…”

Section: ■ Discussionmentioning

confidence: 99%

Mapping a Ketosynthase:Acyl Carrier Protein Binding Interface via Unnatural Amino Acid-Mediated Photo-Cross-Linking

2014

Biochemistry

Probing and interrogating protein interactions that involve acyl carrier proteins (ACP's) in fatty acid synthases and polyketide synthases are critical to understanding the molecular basis for the programmed assembly of complex natural products. Here, we have used unnatural amino acid mutagenesis to site specifically install photo-cross-linking functionality into acyl carrier proteins from diverse systems and the ketosynthase FabF from the Escherichia coli type II fatty acid synthase. Subsequently, a photo-cross-linking assay was employed to systematically probe the ability of FabF to interact with a broad panel of ACP's, illustrating the expected orthogonality of ACP:FabF interactions and the role of charged residues in helix II of the ACP. In addition, FabF residues involved in the binding interaction with the cognate carrier protein were identified via surface scanning mutagenesis and photo-cross-linking. Furthermore, the ability to install the photo-cross-linking amino acid at virtually any position allowed interrogation of the role that carrier protein acylation plays in determining the binding interface with FabF. A conserved carrier protein motif that includes the phosphopantetheinylation site was also shown to play an integral role in maintenance of the AcpP:FabF binding interaction. Our results provide unprecedented insight into the molecular details that describe the AcpP:FabF binding interface and demonstrate that unnatural amino acid based photo-cross-linking is a powerful tool for probing and interrogating protein interactions in complex biosynthetic systems.

“…For example, unnatural phosphopantetheine analogues modified with chemical handles for crosslinking have been successfully used to investigate ACP protein-protein interactions to better understand the protein interactions involved with chain elongation ( Figure 3a ) [141,143]. The efficiency of mechanism-based crosslinking to the KS usually correlates with the strength of the ACP:KS interaction [142]. Moreover, this approach has been used to identify key interactions and set the stage for investigating the utility of such interfaces for combinatorial biosynthesis.…”

Section: Structural and Mechanistic Studies: Implications For Assemblmentioning

confidence: 99%

Harnessing natural product assembly lines: structure, promiscuity, and engineering

Ladner

Journal of Industrial Microbiology and Biotechnology

2016

Many therapeutically relevant natural products are biosynthesized by the action of giant mega-enzyme assembly lines. By leveraging the specificity, promiscuity, and modularity of assembly lines, a variety of strategies have been developed that enable the biosynthesis of modified natural products. This review briefly summarizes recent structural advances related to natural product assembly lines, discusses chemical approaches to probing assembly line structures in the absence of traditional biophysical data, and surveys efforts that harness the inherent or engineered promiscuity of assembly lines for the synthesis of non-natural polyketides and nonribosomal peptide analogues.