Asymmetric Ene-Reduction of α,β-Unsaturated Compounds by F₄₂₀-Dependent Oxidoreductases A Enzymes from Mycobacterium smegmatis

The stereoselective reduction of alkenes conjugated to electron-withdrawing groups by ene-reductases has been extensively applied to the commercial preparation of fine chemicals. Although several different enzyme families are known to possess ene–reductase activity, the old yellow enzyme (OYE) family has been the most thoroughly investigated. Recently, it was shown that a subset of ene-reductases belonging to the flavin/deazaflavin oxidoreductase (FDOR) superfamily exhibit enantioselectivity that is generally complementary to that seen in the OYE family. These enzymes belong to one of several FDOR subgroups that use the unusual deazaflavin cofactor F420. Here, we explore several enzymes of the FDOR-A subgroup, characterizing their substrate range and enantioselectivity with 20 different compounds, identifying enzymes (MSMEG_2027 and MSMEG_2850) that could reduce a wide range of compounds stereoselectively. For example, MSMEG_2027 catalyzed the complete conversion of both isomers of citral to (R)-citronellal with 99% ee, while MSMEG_2850 catalyzed complete conversion of ketoisophorone to (S)-levodione with 99% ee. Protein crystallography combined with computational docking has allowed the observed stereoselectivity to be mechanistically rationalized for two enzymes. These findings add further support for the FDOR and OYE families of ene-reductases displaying general stereocomplementarity to each other and highlight their potential value in asymmetric ene-reduction

Synthesis of a D‑Ring Isomer of Galanthamine via a Radical-Based Smiles Rearrangement Reaction

The 1,9-ethanoiminomethano-bridged tetra­hydro­di­benzo­[<i>b</i>,<i>d</i>]-furan <b>2</b>, a non-natural isomer of the alkaloid (−)-galanthamine (<b>1</b>) varying in the manner in which the D-ring is annulated to the ABC-core, has been prepared in racemic form. The synthetic sequence starts with the cyclopropane <b>3</b> and involves intramolecular Heck alkenylation and radical-based Smiles rearrangement reactions as key steps. Unlike natural product <b>1</b>, but as predicted by docking studies, compound <b>2</b> is not a potent inhibitor of acetylcholine esterase

Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects

How remote mutations can lead to changes in enzyme function at a molecular level is a central question in evolutionary biochemistry and biophysics. Here, we combine laboratory evolution with biochemical, structural, genetic, and computational analysis to dissect the molecular basis for the functional optimization of phosphotriesterase activity in a bacterial lactonase (AiiA) from the metallo-β-lactamase (MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase activity is caused by a more favorable catalytic binding position of the paraoxon substrate in the evolved enzyme that resulted from conformational tinkering of the active site through peripheral mutations. A nonmutated active site residue, Phe68, was displaced by ∼3 Å through the indirect effects of two second-shell trajectory mutations, allowing molecular interactions between the residue and paraoxon. Comparative mutational scanning, i.e., examining the effects of alanine mutagenesis on different genetic backgrounds, revealed significant changes in the functional roles of Phe68 and other nonmutated active site residues caused by the indirect effects of trajectory mutations. Our work provides a quantitative measurement of the impact of second-shell mutations on the catalytic contributions of nonmutated residues and unveils the underlying intramolecular network of strong epistatic mutational relationships between active site residues and more remote residues. Defining these long-range conformational and functional epistatic relationships has allowed us to better understand the subtle, but cumulatively significant, role of second- and third-shell mutations in evolution

Brønsted plot of leaving group pKa values <i>vs</i> log(<i>k</i>_cat/<i>K</i>_M) for a range of substates.

<p>The p<i>K</i>_a values of the leaving groups 2,6-difluoro-4-nitrophenol; quinoxalin-2-ol; 2-fluoro-4-nitrophenol; 2-isopropyl-6-methylpyrimidin-4-ol; 3-fluoro-4-nitrophenol; 4-nitrophenol; 4-hydroxybenzaldehyde; 2,2-dichloroethylenol; 4-hydroxybenzonitrile; 1-(4-hydroxyphenol)ethanone; methyl 4-hydroxybenzoate; 4-hydroxybezamide; 3-chloro-7-hydroxy-4-methyl-2H-chromen-2-one; 2-(ethylthio)ethanethiol; 2-(diethylamino)ethanethiol; 2-(diisopropylamino)ethanethiol; and 4-(methoxymethyl)phenol plotted (left to right) against their log(<i>k</i>_cat/<i>K</i>_M) values. p<i>K</i>_a values were as published elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Caldwell1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Jackson4" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Jackson7" target="_blank">[20]</a> or as calculated using the SPARC online p<i>K</i>_a calculator (<a href="http://ibmlc2.chem.uga.edu/sparc/" target="_blank">http://ibmlc2.chem.uga.edu/sparc/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Hilal1" target="_blank">[38]</a>. The biphasic dependence of the enzyme on p<i>K</i>_a as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Caldwell1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Jackson7" target="_blank">[20]</a> is shown: the curve flattens below a p<i>K</i>_a of ∼8.0 and there is a linear dependence on p<i>K</i>_a at values below <i>ca</i>. 8.0.</p

Roles of Amino Acids at Positions 328 and 331 in AtzA and TriA.

<p>In TriA (A) Cys331 donates a proton to the NH₂− leaving group of melamine and abstracts a proton from Asp328. In AtzA (B), the serine hydroxyl group stabilizes the halide of atrazine in the transition state via a hydrogen bonding interaction and is in turn stabilized by Asn328.</p

Reaction schemes for melamine deaminase (TriA) and atrazine dechlorinase (AtzA).

<p>The hydrolytic deamination of melamine to ammeline by TriA and the dechlorination of atrazine to 2-hydroxyatrazine by AtzA are shown. TriA also possesses a low level of atrazine dechlorinase activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone.0039822-Seffernick2" target="_blank">[17]</a>.</p

Kinetic parameters of purified PTE<i>_Ar</i> and most active variant against malathion for a range of OP insecticides.

<p>Standard deviations for the <i>k</i>_cat and <i>K</i>_M values are given in parentheses below the mean values obtained for triplicate experiments.</p

Hydrolytic activity of PTE<i>_Ar</i>.

<p>A) Schematic showing the PTE<i>_Ar</i>-mediated hydrolysis of malathion. B) Structure of the OP insecticides demeton, chlorpyrifos, parathion and diazinon.</p

Partial step-wise laboratory evolution of TriA to AtzA.

<p>Circles indicate the variants for which the <i>k</i>_cat/<i>K</i>_M values (s⁻¹.M⁻¹; values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone-0039822-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone.0039822.s003" target="_blank">Table S2</a>) for atrazine dechlorination and melamine deamination were determined, and are color coded as follows: TriA, red; generation 1, orange; generation 2, green; generation 3, blue; AtzA, violet. Lines are used to link variants differing by one substitution (thick lines link optimal variants; thin lines link the optimal variants to suboptimal variants – suboptimal variants were not used to generate subsequent variants). Amino acid substitutions discussed in the text have been labelled for clarity, as have the wild-type AtzA and TriA enzymes.</p

Trade-off between atrazine dechlorinase and ametryn hydrolase activity during the transition between AtzA and TriA.

<p>Circles indicate the variants for which the <i>k</i>_cat/<i>K</i>_M values (s⁻¹.M⁻¹; values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone-0039822-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone.0039822.s003" target="_blank">Table S2</a>) for atrazine dechlorination and ametryn hydrolysis are shown. Lines are used to link variants differing by a single amino acid. Each variant has been assigned a letter and the identity of the each variant’s direct parent is indicated together with the distinguishing amino acid substitution. The letter assignments correspond to those found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone-0039822-g003" target="_blank">Fig. 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone-0039822-t001" target="_blank">Table 1</a>.</p