Connecting the geometric and electronic structures of the nitrogenase iron-molybdenum cofactor through site-selective 57Fe labelling.

Understanding the chemical bonding in the catalytic cofactor of the Mo nitrogenase (FeMo-co) is foundational for building a mechanistic picture of biological nitrogen fixation. A persistent obstacle towards this goal has been that the 57Fe-based spectroscopic data-although rich with information-combines responses from all seven Fe sites, and it has therefore not been possible to map individual spectroscopic responses to specific sites in the three-dimensional structure. Here we have addressed this challenge by incorporating 57Fe into a single site of FeMo-co. Spectroscopic analysis of the resting state informed on the local electronic structure of the terminal Fe1 site, including its oxidation state and spin orientation, and, in turn, on the spin-coupling scheme for the entire cluster. The oxidized resting state and the first intermediate in nitrogen fixation were also characterized, and comparisons with the resting state provided molecular-level insights into the redox chemistry of FeMo-co.

Postbiosynthetic modification of a precursor to the nitrogenase iron-molybdenum cofactor.

Nitrogenases utilize Fe-S clusters to reduce N2 to NH3 The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their electronic structures, mechanisms, and biosyntheses. To facilitate their spectroscopic analysis, we are developing methods for incorporating 57Fe into specific sites of nitrogenase cofactors, and we report herein site-selective 57Fe labeling of the L-cluster-a carbide-containing, [Fe8S9C] precursor to the Mo nitrogenase catalytic cofactor. Treatment of the isolated L-cluster with the chelator ethylenediaminetetraacetate followed by reconstitution with 57Fe2+ results in 57Fe labeling of the terminal Fe sites in high yield and with high selectivity. This protocol enables the generation of L-cluster samples in which either the two terminal or the six belt Fe sites are selectively labeled with 57Fe. Mössbauer spectroscopic analysis of these samples bound to the nitrogenase maturase Azotobacter vinelandii NifX reveals differences in the primary coordination sphere of the terminal Fe sites and that one of the terminal sites of the L-cluster binds to H35 of Av NifX. This work provides molecular-level insights into the electronic structure and biosynthesis of the L-cluster and introduces postbiosynthetic modification as a promising strategy for studies of nitrogenase cofactors.

Using Peptide Mimics to Study the Biosynthesis of the Side-Ring System of Nosiheptide.

Thiopeptide natural products have gained interest recently for their diverse pharmacological properties, including antibacterial, antifungal, anticancer, and antimalarial activities. Due to their inherent poor solubility and uptake, there is interest in developing new thiopeptides that mimic these unique structures, but which exhibit better pharmacokinetic properties. One strategy is to exploit the biosynthetic pathways using a chemoenzymatic approach to make analogs. However, a complete understanding of thiopeptide biosynthesis is not available, especially for those molecules that contain a large number of modifications to the thiopeptide core. This gap in knowledge and the lack of a facile method for generating a variety of thiopeptide intermediates makes studying particular enzymatic steps difficult. We developed a method to produce thiopeptide mimics based on established synthetic procedures to study the reaction catalyzed by NosN, the class C radical S-adenosylmethionine methylase involved in carbon transfer to C4 of 3-methylindolic acid and completion of the side-ring system in nosiheptide. Herein, we detail strategies for overproducing and isolating NosN, as well as procedures for synthesizing substrate mimics to study the formation of the side-ring system of nosiheptide.

NosN, a Radical S-Adenosylmethionine Methylase, Catalyzes Both C1 Transfer and Formation of the Ester Linkage of the Side-Ring System during the Biosynthesis of Nosiheptide.

Nosiheptide, a member of the e series of macrocyclic thiopeptide natural products, contains a side-ring system composed of a 3,4-dimethylindolic acid (DMIA) moiety connected to Glu6 and Cys8 of the thiopeptide backbone via ester and thioester linkages, respectively. Herein, we show that NosN, a predicted class C radical S-adenosylmethionine (SAM) methylase, catalyzes both the transfer of a C1 unit from SAM to 3-methylindolic acid linked to Cys8 of a synthetic substrate surrogate as well as the formation of the ester linkage between Glu6 and the nascent C4 methylene moiety of DMIA. In contrast to previous studies that indicated that 5'-methylthioadenosine is the immediate methyl donor in the reaction, in our studies, SAM itself plays this role, giving rise to S-adenosylhomocysteine as a coproduct of the reaction.

Rerouting the Pathway for the Biosynthesis of the Side Ring System of Nosiheptide: The Roles of NosI, NosJ, and NosK.

Nosiheptide (NOS) is a highly modified thiopeptide antibiotic that displays formidable in vitro activity against a variety of Gram-positive bacteria. In addition to a central hydroxypyridine ring, NOS contains several other modifications, including multiple thiazole rings, dehydro-amino acids, and a 3,4-dimethylindolic acid (DMIA) moiety. The DMIA moiety is required for NOS efficacy and is synthesized from l-tryptophan in a series of reactions that have not been fully elucidated. Herein, we describe the role of NosJ, the product of an unannotated gene in the biosynthetic operon for NOS, as an acyl carrier protein that delivers 3-methylindolic acid (MIA) to NosK. We also reassign the role of NosI as the enzyme responsible for catalyzing the ATP-dependent activation of MIA and MIA's attachment to the phosphopantetheine moiety of NosJ. Lastly, NosK catalyzes the transfer of the MIA group from NosJ-MIA to a conserved serine residue (Ser102) on NosK. The X-ray crystal structure of NosK, solved to 2.3 Å resolution, reveals that the protein is an α/ß-fold hydrolase. Ser102 interacts with Glu210 and His234 to form a catalytic triad located at the bottom of an open cleft that is large enough to accommodate the thiopeptide framework.