Mechanism of Oxidative Ring‐Closure as Part of the Hygromycin Biosynthesis Step by a Nonheme Iron Dioxygenase

Abstract

Nonheme iron dioxygenases catalyze vital reactions for biosystems including the biosynthesis of antibiotics. One such enzyme, namely the hygromycin biosynthesis enzyme (HygX), performs an oxidative ring-closure reaction to form an orthoÀ ester product, which is a relevant reaction step for drug synthesis and biotechnology. To understand the selective reaction mechanism of oxidative ring-closure to form orthoÀ ester products in HygX, we investigated its catalytic reaction mechanism leading to various products. Large active site cluster models were set-up and various pathways for substrate activation have been calculated. The work identifies a high-valent iron(IV)-oxo species in the quintet spin state as the active oxidant that selectively abstracts a proton of an alcohol group of the substrate, which is followed by a hydrogen atom abstraction from a tertiary CÀ H group and rapid electron transfer. The latter-formed biradical intermediate rearranges to form the desaturated ring-closed product. The calculations show that an active site Lys residue donates positive charge to the metalÀ oxo group and guides the reaction to a chemoselective desaturation pathway.

Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1

“…In an alternative pathway, the Fe III −OH species may undergo the OH‐rebound to the C21 radical site via a slightly larger barrier of 5.2 kcal mol −1 , affording to the hydroxylated substrate which is 27.6 kcal mol −1 more stable than the intermediate IC1 (Figure S13). The calculated rebound barrier is in line with previous studies [60–73] . The hydroxylated species would lead to the side product 8 through elimination (refer to Scheme 3), as observed in experiments.…”

“…Thec alculated rebound barrier is in line with previous studies. [60][61][62][63][64][65][66][67][68][69][70][71][72][73] The hydroxylated species would lead to the side product 8 through elimination (refer to Scheme 3), as observed in experiments. Clearly,t he dioxygen attack is kinetically favorable over the rebound pathway,w hile the rebound pathway is more thermodynamically favorable.Inthe situation of low concen- S3 and Figure S15).…”

pH Changes That Induce an Axial Ligand Effect on Nonheme Iron(IV) Oxo Complexes with an Appended Aminopropyl Functionality

“…Hence, the differences in reactivity seem to occur through charge interaction of a positively charged amine group along the Fe–O–substrate axis. This is in line with recent computational studies on the hygromycin biosynthesis enzyme that has an active site Lys residue that assists in the chemical reaction mechanism . In addition, the value of the imaginary frequency is very close; therefore, the potential energy surfaces will be very similar.…”

Density Functional Theory Study into the Reaction Mechanism of Isonitrile Biosynthesis by the Nonheme Iron Enzyme ScoE

“…As such, a charged residue in the substrate binding pocket and particularly a positively charged residue that is hydrogen bonding to the oxo group has a major influence on the hydrogen atom abstraction barrier. This is in analogy to the hygromycin biosynthesis enzyme, where an active site Lys residue was shown to affect rate constants and reaction selectivities by guiding the reaction to a specific product channel [99]. Furthermore, work on the hectochlorin biosynthesis enzyme identified an active site Glu residue that was shown to donate negative charge to an iron-bound halide and influence the regioselectivity of halogenation over hydroxylation in a nonheme iron enzyme by polarizing the Fe-Cl bond better [98].…”

“…Clearly, the protein induces local perturbations to the active site pocket that change the relative C-H and N-H bond strengths. Previous work on nonheme iron dioxygenases showed that charged residues in the substrate binding pocket and active site, such as Lys residues, can donate positive charge into the binding pocket and affect local bonds strengths and selectivities dramatically [68,[97][98][99]. Moreover, studies using electric field effects showed that these external perturbations can influence electronic distributions as well as chemoselectivities of substrate activation by enzymes [100][101][102].…”