Constitutively Active Glutaminase Variants Provide Insights into the Activation Mechanism of Anthranilate Synthase

The glutamine amidotransferase (GATase) family comprises enzyme complexes which consist of glutaminase and synthase subunits that catalyze in a concerted reaction the incorporation of nitrogen within various metabolic pathways. An important feature of GATases is the strong stimulation of glutaminase activity by the associated synthase. To understand the mechanism of this tight activity regulation, we probed by site-directed mutagenesis four residues of the glutaminase subunit TrpG from anthranilate synthase that are located between the catalytic Cys–His–Glu triad and the synthase subunit TrpE. In order to minimize structural perturbations induced by the introduced exchanges, the amino acids from TrpG were substituted with the corresponding residues of the closely related glutaminase HisH from imidazole glycerol phosphate synthase. Steady-state kinetic characterization showed that, in contrast to wild-type TrpG, two TrpG variants with single exchanges constitutively hydrolyzed glutamine in the absence of TrpE. A reaction assay performed with hydroxylamine as a stronger nucleophile replacing water and a filter assay with radiolabeled glutamine indicated that the formation of the thioester intermediate is the rate-limiting step of constitutive glutamine hydrolysis. Molecular dynamics simulations with wild-type TrpG and constitutively active TrpG variants suggest that the introduced amino acid exchanges result in a distance reduction between the active site Cys–His pair, which facilitates the deprotonation of the sulfhydryl group of the catalytic cysteine and thus enables its nucleophilic attack onto the carboxamide group of the glutamine side chain. We propose that native TrpG in the anthranilate synthase complex is activated by a similar mechanism

Conservation of the Folding Mechanism between Designed Primordial (βα)₈-Barrel Proteins and Their Modern Descendant

The (βα)₈-barrel is among the most ancient, frequent, and versatile enzyme structures. It was proposed that modern (βα)₈-barrel proteins have evolved from an ancestral (βα)₄-half-barrel by gene duplication and fusion. We explored whether the mechanism of protein folding has remained conserved during this long-lasting evolutionary process. For this purpose, potential primordial (βα)₈-barrel proteins were constructed by the duplication of a (βα)₄ element of a modern (βα)₈-barrel protein, imidazole glycerol phosphate synthase (HisF), followed by the optimization of the initial construct. The symmetric variant Sym1 was less stable than HisF and its crystal structure showed disorder in the contact regions between the half-barrels. The next generation variant Sym2 was more stable than HisF, and the contact regions were well resolved. Remarkably, both artificial (βα)₈-barrels show the same refolding mechanism as HisF and other modern (βα)₈-barrel proteins. Early in folding, they all equilibrate rapidly with an off-pathway species. On the productive folding path, they form closely related intermediates and reach the folded state with almost identical rates. The high energy barrier that synchronizes folding is thus conserved. The strong differences in stability between these proteins develop only after this barrier and lead to major changes in the unfolding rates. We conclude that the refolding mechanism of (βα)₈-barrel proteins is robust. It evolved early and, apparently, has remained conserved upon the diversification of sequences and functions that have taken place within this large protein family

Structural and Computational Insight into the Catalytic Mechanism of Limonene Epoxide Hydrolase Mutants in Stereoselective Transformations

Directed evolution of limonene epoxide hydrolase (LEH), which catalyzes the hydrolytic desymmetrization reactions of cyclopentene oxide and cyclohexene oxide, results in (<i>R,R</i>)- and (<i>S,S</i>)-selective mutants. Their crystal structures combined with extensive theoretical computations shed light on the mechanistic intricacies of this widely used enzyme. From the computed activation energies of various pathways, we discover the underlying stereochemistry for favorable reactions. Surprisingly, some of the most enantioselective mutants that rapidly convert cyclohexene oxide do not catalyze the analogous transformation of the structurally similar cyclopentene oxide, as shown by additional X-ray structures of the variants harboring this slightly smaller substrate. We explain this puzzling observation on the basis of computational calculations which reveal a disrupted alignment between nucleophilic water and cyclopentene oxide due to the pronounced flexibility of the binding pocket. In contrast, in the stereoselective reactions of cyclohexene oxide, reactive conformations are easily reached. The unique combination of structural and computational data allows insight into mechanistic details of this epoxide hydrolase and provides guidance for future protein engineering in reactions of structurally different substrates

Structural and Computational Insight into the Catalytic Mechanism of Limonene Epoxide Hydrolase Mutants in Stereoselective Transformations

Directed evolution of limonene epoxide hydrolase (LEH), which catalyzes the hydrolytic desymmetrization reactions of cyclopentene oxide and cyclohexene oxide, results in (<i>R,R</i>)- and (<i>S,S</i>)-selective mutants. Their crystal structures combined with extensive theoretical computations shed light on the mechanistic intricacies of this widely used enzyme. From the computed activation energies of various pathways, we discover the underlying stereochemistry for favorable reactions. Surprisingly, some of the most enantioselective mutants that rapidly convert cyclohexene oxide do not catalyze the analogous transformation of the structurally similar cyclopentene oxide, as shown by additional X-ray structures of the variants harboring this slightly smaller substrate. We explain this puzzling observation on the basis of computational calculations which reveal a disrupted alignment between nucleophilic water and cyclopentene oxide due to the pronounced flexibility of the binding pocket. In contrast, in the stereoselective reactions of cyclohexene oxide, reactive conformations are easily reached. The unique combination of structural and computational data allows insight into mechanistic details of this epoxide hydrolase and provides guidance for future protein engineering in reactions of structurally different substrates

Cavity Size Engineering of a β‑Barrel Protein Generates Efficient Biohybrid Catalysts for Olefin Metathesis

Incorporation of a synthetic metal catalyst into a protein scaffold yields a biohybrid catalyst, with a remarkable performance in aqueous media and the broad reaction scope of organometallic catalysts. A major challenge for efficient catalysis is the design of the interface between the protein scaffold and the metal catalyst. Until now, protein scaffolds have primarily been engineered by exchanging individual amino acids to anchor metal catalysts and alter their immediate environment. Here, cavity size engineering of the β-barrel protein nitrobindin was performed by duplicating multiple β-strands to generate an expanded variant. The approach of cavity size engineering enabled covalent incorporation of bulky catalysts at excellent coupling efficiencies and yielded excellent conversions in olefin metathesis, including ring-closing metathesis, ring-opening metathesis polymerization, and cross metathesis (conversions up to 99% and turnover numbers up to 10000)