Kinetic Analysis and Probing with Substrate Analogues of the Reaction Pathway of the Nitrile Reductase QueF from Escherichia coli.

The enzyme QueF catalyzes a four-electron reduction of a nitrile group into an amine, the only reaction of this kind known in biology. In nature, QueF converts 7-cyano-7-deazaguanine (preQ0) into 7-aminomethyl-7-deazaguanine (preQ1) for the biosynthesis of the tRNA-inserted nucleoside queuosine. The proposed QueF mechanism involves a covalent thioimide adduct between preQ0 and a cysteine nucleophile in the enzyme, and this adduct is subsequently converted into preQ1 in two NADPH-dependent reduction steps. Here, we show that the Escherichia coli QueF binds preQ0 in a strongly exothermic process (ΔH = -80.3 kJ/mol; -TΔS = 37.9 kJ/mol, Kd = 39 nm) whereby the thioimide adduct is formed with half-of-the-sites reactivity in the homodimeric enzyme. Both steps of preQ0 reduction involve transfer of the 4-pro-R-hydrogen from NADPH. They proceed about 4-7-fold more slowly than trapping of the enzyme-bound preQ0 as covalent thioimide (1.63 s-1) and are thus mainly rate-limiting for the enzyme's kcat (=0.12 s-1). Kinetic studies combined with simulation reveal a large primary deuterium kinetic isotope effect of 3.3 on the covalent thioimide reduction and a smaller kinetic isotope effect of 1.8 on the imine reduction to preQ1 7-Formyl-7-deazaguanine, a carbonyl analogue of the imine intermediate, was synthesized chemically and is shown to be recognized by QueF as weak ligand for binding (ΔH = -2.3 kJ/mol; -TΔS = -19.5 kJ/mol) but not as substrate for reduction or oxidation. A model of QueF substrate recognition and a catalytic pathway for the enzyme are proposed based on these data.

An investigation of nitrile transforming enzymes in the chemo-enzymatic synthesis of the taxol sidechain.

Paclitaxel (taxol) is an antimicrotubule agent widely used in the treatment of cancer. Taxol is prepared in a semisynthetic route by coupling the N-benzoyl-(2R,3S)-3-phenylisoserine sidechain to the baccatin III core structure. Precursors of the taxol sidechain have previously been prepared in chemoenzymatic approaches using acylases, lipases, and reductases, mostly featuring the enantioselective, enzymatic step early in the reaction pathway. Here, nitrile hydrolysing enzymes, namely nitrile hydratases and nitrilases, are investigated for the enzymatic hydrolysis of two different sidechain precursors. Both sidechain precursors, an openchain α-hydroxy-ß-amino nitrile and a cyanodihydrooxazole, are suitable for coupling to baccatin III directly after the enzymatic step. An extensive set of nitrilases and nitrile hydratases was screened towards their activity and selectivity in the hydrolysis of two taxol sidechain precursors and their epimers. A number of nitrilases and nitrile hydratases converted both sidechain precursors and their epimers.

Targeting the substrate binding site of E. coli nitrile reductase QueF by modeling, substrate and enzyme engineering.

Nitrile reductase QueF catalyzes the reduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ0) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ1) in the biosynthetic pathway of the hypermodified nucleoside queuosine. It is the only enzyme known to catalyze a reduction of a nitrile to its corresponding primary amine and could therefore expand the toolbox of biocatalytic reactions of nitriles. To evaluate this new oxidoreductase for application in biocatalytic reactions, investigation of its substrate scope is prerequisite. We report here an investigation of the active site binding properties and the substrate scope of nitrile reductase QueF from Escherichia coli. Screenings with simple nitrile structures revealed high substrate specificity. Consequently, binding interactions of the substrate to the active site were identified based on a new homology model of E. coli QueF and modeled complex structures of the natural and non-natural substrates. Various structural analogues of the natural substrate preQ0 were synthesized and screened with wild-type QueF from E. coli and several active site mutants. Two amino acid residues Cys190 and Asp197 were shown to play an essential role in the catalytic mechanism. Three non-natural substrates were identified and compared to the natural substrate regarding their specific activities by using wild-type and mutant nitrile reductase.

Chemoenzymatic one-pot reaction from carboxylic acid to nitrile via oxime.

We report a new chemoenzymatic cascade starting with aldehyde synthesis by carboxylic acid reductase (CAR) followed by chemical in situ oxime formation. The final step to the nitrile is catalyzed by aldoxime dehydratase (Oxd). Full conversions of phenylacetic acid and hexanoic acid were achieved in a two-phase mode.

Enantioseparation of nonproteinogenic amino acids.

The enantioseparation of structurally related N-protected beta-/gamma-amino acids, beta-/gamma-amino amides, and beta-/gamma-amino nitriles by using six different commercially available chiral stationary phases (CSPs) is reported. The synthetic key step to introduce stereochemical asymmetry into all compounds is an enzymatic kinetic resolution of the racemic nitriles to the respective amino amides and/or amino acids, depending on the class of enzyme (nitrile hydratase or nitrilase) applied. The separation efficiencies of all CSPs with regard to functional groups as well as structural variations of the amino acid derivatives depicted in Fig. 1 are discussed.

Influence of relative configuration of disubstituted cyclopentanes and -hexanes on 13C shifts.

(13)C shifts of disubstituted cyclopentane and cyclohexane derivatives were compared in dependence on the relative configuration of the two substituents. A diequatorial substitution correlates with deshielding compared to other substitution patterns. Some novel fluorinated cyclopentanes and -hexanes including their DFT calculation-assisted structure elucidation are described.

Enzyme stabilizer DTT catalyzes nitrilase analogue hydrolysis of nitriles.

Amides are the dominating products in some nitrilase catalyzed conversions of alpha-activated nitriles, but unexpectedly this hydrolytic reaction is also catalyzed by 1,4-dithio-dl-threitol (DTT), a standard antioxidizing enzyme stabilizer.

Nitrilases catalyze key step to conformationally constrained GABA analogous gamma-amino acids in high optical purity.

Five- and six-membered carbocyclic gamma-amino acids were prepared in high enantiomeric purity by nitrilase-mediated transformation of hitherto unreported gamma-amino nitriles. The nitrilases investigated reveal a strong enantiopreference for cis-isomers (up to 99% ee), whereas trans-isomers were available in up to 86% ee. The biocatalytic enantioselective syntheses of cis-3-aminocyclohexanecarboxylic acid (3b), trans-3-aminocyclohexanecarboxylic acids (4b, 6b, 8b) as well as trans-3-aminocylopentanecarboxylic acid (2b) are hereby reported for the first time.