Ionizable residues and monoatomic ions in solution modulate enzyme catalysis and the structural stability of proteins; however, the delicate interplay between these short-range charges and longrange charges, and their contributions to the electrostatic environment in a protein active site, is currently not fully understood. The study presented here utilizes the FMN-dependent NADH:quinone oxidoreductase from Pseudomonas aeruginosa PAO1 (NQO, EC 1.6.5.9, UniProtKB Q9I4V0) as a model system to study the effect of introducing an active site negative charge on the flavin absorption spectrum both in the absence and presence of a long-range electrostatic potential coming from solution ions. Using pH-dependent UV-visible spectroscopy, there were no observed changes in the flavin absorption spectrum when an active site tyrosine (Y277) deprotonated in vitro. These results could only be reproduced computationally using Average Solvent Electrostatic Configuration (ASEC) hybrid quantum mechanics / molecular mechanics (QM/MM) simulations that included both positive and negative solution ions. The same calculations performed with minimal ions to neutralize protein charges predicted that deprotonating Y277 would significantly affect the flavin absorption spectrum. Analyzing the distribution of solution ions from ASEC and radial distribution functions derived from molecular dynamics indicated that the solution ions reorganize around the protein surface upon Y277 deprotonation to cancel the effect of the tyrosinate on the flavin absorption spectrum. Biochemical experiments were performed to support this hypothesis. This work highlights the importance of salt ions, which are sometimes overlooked, since they can contribute a non-uniform and anisotropic long-range potential to the electrostatic environment of an active site.
Nitration reactions are crucial for many industrial syntheses; however, current protocols lack site specificity and employ hazardous chemicals. The noncanonical cytochrome P450 enzymes RufO and TxtE catalyze the only known direct aromatic nitration reactions in nature, making them attractive model systems for the development of analogous biocatalytic and/or biomimetic reactions that proceed under mild conditions. While the associated mechanism has been well-characterized in TxtE, much less is known about RufO. Herein we present the first structure of RufO alongside a series of computational and biochemical studies investigating its unusual reactivity. We demonstrate that free Ltyrosine is not readily accepted as a substrate despite previous reports to the contrary. Instead, we propose that RufO natively modifies L-tyrosine tethered to the peptidyl carrier protein of a nonribosomal peptide synthetase encoded by the same biosynthetic gene cluster and present both docking and molecular dynamics simulations consistent with this hypothesis. Our results expand the scope of direct enzymatic nitration reactions and provide the first evidence for such a modification of a peptide synthetase-bound substrate. Both of these insights may aid in the downstream development of biocatalytic approaches to synthesize rufomycin analogues and related drug candidates.
Nitration reactions are crucial for many industrial syntheses; however, current protocols lack site-specificity and employ hazardous chemicals. The non-canonical cytochrome P450 enzymes RufO and TxtE catalyze the only known direct aromatic nitration reactions in nature, making them attractive model systems for the development of analogous biocatalytic and/or biomimetic reactions that proceed under mild conditions. While the associated mechanism has been well characterized in TxtE, much less is known about RufO. Herein, we present the first structure of RufO alongside a series of computational and biochemical studies investigating its unusual reactivity. We demonstrate that free L-tyrosine is not readily accepted as a substrate, despite previous reports to the contrary. Instead, we propose that RufO natively modifies L-tyrosine tethered to the peptidyl carrier protein of a non-ribosomal peptide synthetase encoded by the same biosynthetic gene cluster and present both docking and molecular dynamics simulations consistent with this hypothesis. Our results expand the scope of direct enzymatic nitration reactions and provide the first evidence for such a modification of a peptide synthetase-bound substrate that may aid in the downstream development of biocatalytic approaches to synthesize rufomycin analogs and related drug candidates.
Ionizable residues and monoatomic ions in solution modulate enzyme catalysis and the structural stability of proteins; however, the delicate interplay between these short-range charges and long-range charges, and their contributions to the electrostatic environment in a protein active site, is currently not fully understood. The study presented here utilizes the FMN-dependent NADH:quinone oxidoreductase from Pseudomonas aeruginosa PAO1 (NQO, EC 1.6.5.9, UniProtKB Q9I4V0) as a model system to study the effect of introducing an active site negative charge on the flavin absorption spectrum both in the absence and presence of a long-range electrostatic potential coming from solution ions. Using pH-dependent UV-visible spectroscopy, there were no observed changes in the flavin absorption spectrum when an active site tyrosine (Y277) deprotonated in vitro. These results could only be reproduced computationally using Average Solvent Electrostatic Configuration (ASEC) hybrid quantum mechanics / molecular mechanics (QM/MM) simulations that included both positive and negative solution ions. The same calculations performed with minimal ions to neutralize protein charges predicted that deprotonating Y277 would significantly affect the flavin absorption spectrum. Analyzing the distribution of solution ions from ASEC and radial distribution functions derived from molecular dynamics indicated that the solution ions reorganize around the protein surface upon Y277 deprotonation to cancel the effect of the tyrosinate on the flavin absorption spectrum. Biochemical experiments were performed to support this hypothesis. This work highlights the importance of salt ions, which are sometimes overlooked, since they can contribute a non-uniform and anisotropic long-range potential to the electrostatic environment of an active site.
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