Structural basis for precursor protein-directed ribosomal peptide macrocyclization.

Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight into the unique protein-protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.

Structure and noncanonical chemistry of nonribosomal peptide biosynthetic machinery.

Structural biology has provided significant insights into the complex chemistry and macromolecular organization of nonribosomal peptide synthetases. In addition, novel pathways are continually described, expanding the knowledge of known biosynthetic chemistry.

Synthesis and structure confirmation of fuscachelins A and B, structurally unique natural product siderophores from Thermobifida fusca.

The fuscachelin siderophores have been prepared synthetically as have their metal chelation complexes. The heterodimeric nature of the fuscachelin decamer lends itself to a convergent synthetic strategy. Synthetic access to the natural products and intermediates will provide readily adaptable tools in future studies examining iron-sequestration and the biosynthetic machinery.

Probing the structural basis of oxygen binding in a cofactor-independent dioxygenase.

The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.

Oxygen diffusion pathways in a cofactor-independent dioxygenase.

Molecular oxygen plays an important role in a wide variety of enzymatic reactions. Through recent research efforts combining computational and experimental methods a new view of O2 diffusion is emerging, where specific channels guide O2 to the active site. The focus of this work is DpgC, a cofactor-independent oxygenase. Molecular dynamics simulations, together with mutagenesis experiments and xenon-binding data, reveal that O2 reaches the active site of this enzyme using three main pathways and four different access points. These pathways connect a series of dynamic hydrophobic pockets, concentrating O2 at a specific face of the enzyme substrate. Extensive molecular dynamics simulations provide information about which pathways are more frequently used. This data is consistent with the results of kinetic measurements on mutants and is difficult to obtain using computational cavity-location methods. Taken together, our results reveal that although DpgC is rare in its ability of activating O2 in the absence of cofactors or metals, the way O2 reaches the active site is similar to that reported for other O2-using proteins: multiple access channels are available, and the architecture of the pathway network can provide regio- and stereoselectivity. Our results point to the existence of common themes in O2 access that are conserved among very different types of proteins.

Structure guided approaches toward exploiting and manipulating nonribosomal peptide and polyketide biosynthetic pathways.

Nonribosomal peptide and polyketide natural products are structurally diverse small molecules synthesized on complex enzyme assemblies. The ability to rationally engineer secondary metabolic pathways is a promising approach to novel therapeutics. Atomic resolution structures of biosynthetic enzymes provide information on active site architecture and macromolecular assembly that can aid in the engineering of new compounds. This review surveys recent applications toward biosynthetic engineering of natural products guided by structural biology.