David Mansell

A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity.

We have conducted a site‐specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,β‐unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2‐phenyl‐1‐nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to CC reduction. These mutants showed an enhancement in a minor side reaction and formed 2‐phenylpropanal oxime from 2‐phenyl‐1‐nitropropene. The multiple binding conformations of hydroxy substituted nitro‐olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.

Focused Directed Evolution of Pentaerythritol Tetranitrate Reductase by Using Automated Anaerobic Kinetic Screening of Site-Saturated Libraries

This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96‐well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site‐saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)‐2‐aryl‐1‐nitropropene and α‐methyl‐trans‐cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,β‐unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.

Biocatalytic asymmetric alkene reduction: crystal structure and characterization of a double bond reductase from Nicotiana tabacum

The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of “-ene” reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover “-ene” reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B4 dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of α,β-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE “-ene” reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP+ and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme.

Chelatable iron pool: inositol 1,2,3-trisphosphate fulfils the conditions required to be a safe cellular iron ligand.

Mammalian cells contain a pool of iron that is not strongly bound to proteins, which can be detected with fluorescent chelating probes. The cellular ligands of this biologically important “chelatable”, “labile” or “transit” iron are not known. Proposed ligands are problematic, because they are saturated by magnesium under cellular conditions and/or because they are not “safe”, i.e. they allow iron to catalyse hydroxyl radical formation. Among small cellular molecules, certain inositol phosphates (InsPs) excel at complexing Fe3+ in such a “safe” manner in vitro. However, we previously calculated that the most abundant InsP, inositol hexakisphosphate, cannot interact with Fe3+ in the presence of cellular concentrations of Mg2+. In this work, we study the metal complexation behaviour of inositol 1,2,3-trisphosphate [Ins(1,2,3)P3], a cellular constituent of unknown function and the simplest InsP to display high-affinity, “safe”, iron complexation. We report thermodynamic constants for the interaction of Ins(1,2,3)P3 with Na+, K+, Mg2+, Ca2+, Cu2+, Fe2+ and Fe3+. Our calculations indicate that Ins(1,2,3)P3 can be expected to complex all available Fe3+ in a quantitative, 1:1 reaction, both in cytosol/nucleus and in acidic compartments, in which an important labile iron subpool is thought to exist. In addition, we calculate that the fluorescent iron probe calcein would strip Fe3+ from Ins(1,2,3)P3 under cellular conditions, and hence labile iron detected using this probe may include iron bound to Ins(1,2,3)P3. Therefore Ins(1,2,3)P3 is the first viable proposal for a transit iron ligand.

Electro-enzymatic viologen-mediated substrate reduction using pentaerythritol tetranitrate reductase and a parallel, segmented fluid flow system

Many redox enzymes require expensive reduced cofactors like NAD(P)H which need to be recycled during catalysis, presenting a major cost and technical barrier to industrial exploitation. An electrochemical biphasic microfluidic setup is presented here, in which these cofactors are replaced by a mediator (methyl viologen) that acts by feeding electrons into the active site of the enzyme pentaerythritol tetranitrate reductase (PETNR). In this microfluidic recirculation setup, both enzyme and mediator remain in the reactor for reuse, allowing easy product recovery. System optimisation studies were performed using 2-cyclohexen-1-one as a model substrate prior to the investigation of a variety of different substrates whose reduction rates were determined to be 15–70% of those obtained when NADPH was used as sole electron donor. Additional data obtained with a thermophilic ‘ene’ reductase (TOYE) support the potential universality of this device for possible industrial applications.

Light-driven biocatalytic reduction of α,β-unsaturated compounds by ene reductases employing transition metal complexes as photosensitizers

Efficient and cost effective nicotinamide cofactor regeneration is essential for industrial-scale bio-hydrogenations employing flavin-containing biocatalysts such as the Old Yellow Enzymes.

A surprising observation that oxygen can affect the product enantiopurity of an enzyme‐catalysed reaction

Enzymes are natural catalysts, controlling reactions with typically high stereospecificity and enantiospecificity in substrate selection and/or product formation. This makes them useful in the synthesis of industrially relevant compounds, particularly where highly enantiopure products are required. The flavoprotein pentaerythritol tetranitrate (PETN) reductase is a member of the Old Yellow Enzyme family, and catalyses the asymmetric reduction of β‐alkyl‐β‐arylnitroalkenes. Under aerobic conditions, it additionally undergoes futile cycles of NAD(P)H reduction of flavin, followed by reoxidation by oxygen, which generates the reactive oxygen species (ROS) hydrogen peroxide and superoxide. Prior studies have shown that not all reactions catalysed by PETN reductase yield enantiopure products, such as the reduction of (E)‐2‐phenyl‐1‐nitroprop‐1‐ene (PNE) to produce (S)‐2‐phenyl‐1‐nitropropane (PNA) with variable enantiomeric excess (ee). Recent independent studies of (E)‐PNE reduction by PETN reductase showed that the major product formed could be switched to (R)‐PNA, depending on the reaction conditions. We investigated this phenomenon, and found that the presence of oxygen and ROS influenced the overall product enantiopurity. Anaerobic reactions produced consistently higher nitroalkane (S)‐PNA product yields than aerobic reactions (64% versus 28%). The presence of oxygen dramatically increased the preference for (R)‐PNA formation (up to 52% ee). Conversely, the presence of the ROS superoxide and hydrogen peroxide switched the preference to (S)‐PNA product formation. Given that oxygen has no role in the natural catalytic cycle, these findings demonstrate a remarkable ability to manipulate product enantiopurity of this enzyme‐catalysed reaction by simple manipulation of reaction conditions. Potential mechanisms of this unusual behaviour are discussed.

Mechanistic Reappraisal of Early Stage Photochemistry in the Light-Driven Enzyme Protochlorophyllide Oxidoreductase

The light-driven enzyme protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This reaction is a key step in the biosynthesis of chlorophyll. Ultrafast photochemical processes within the Pchlide molecule are required for catalysis and previous studies have suggested that a short-lived excited-state species, known as I675*, is the first catalytic intermediate in the reaction and is essential for capturing excitation energy to drive subsequent hydride and proton transfers. The chemical nature of the I675* excited state species and its role in catalysis are not known. Here, we report time-resolved pump-probe spectroscopy measurements to study the involvement of the I675* intermediate in POR photochemistry. We show that I675* is not unique to the POR-catalyzed photoreduction of Pchlide as it is also formed in the absence of the POR enzyme. The I675* species is only produced in samples that contain both Pchlide substrate and Chlide product and its formation is dependent on the pump excitation wavelength. The rate of formation and the quantum yield is maximized in 50∶50 mixtures of the two pigments (Pchlide and Chlide) and is caused by direct energy transfer between Pchlide and neighboring Chlide molecules, which is inhibited in the polar solvent methanol. Consequently, we have re-evaluated the mechanism for early stage photochemistry in the light-driven reduction of Pchlide and propose that I675* represents an excited state species formed in Pchlide-Chlide dimers, possibly an excimer. Contrary to previous reports, we conclude that this excited state species has no direct mechanistic relevance to the POR-catalyzed reduction of Pchlide.

7.11 Reduction: Enantioselective Bioreduction of C–C Double Bonds

Biocatalytic asymmetric activated alkene reduction by whole-cell suspensions and enzymes has been the subject of considerable research due to the often high efficiency and regio-, stereo-, and enantioselectivity of the reaction. Reactions catalyzed by enzymes are most often flavin-containing NAD(P)H-dependent enzymes from the Old Yellow Enzyme Family. These enzymes reduce the CC bond of activated aldehydes, ketones, nitroalkenes, and carboxylic acids to produce a variety of industrially useful compounds. Bakers yeast and other microorganisms have been used extensively as robust, versatile whole-cell biocatalysts for activated alkene reduction and contain constitutive enzymes for coenzyme recycling. However, secondary reductions often occur, such as ketone reduction, due to the presence of other oxidoreductases. The use of isolated enzymes reduces the likelihood of side product formation, but requires the addition of a coenzyme-recycling system. This chapter will summarize the current knowledge of biocatalytic alkene reduction catalyzed by a wide range of microorganisms and isolated enzymes.

Conformational Evaluation of Indol-3-yl-N-alkyl-glyoxalylamides and Indol-3-yl-N,N-dialkyl-glyoxalylamides

ABSTRACT Restricted rotation in indol-3-yl-N-alkyl- and indol-3-yl-N,N-dialkyl-glyoxalylamides can in principle give the syn-periplanar and anti-periplanar rotamers. In asymmetrically disubstituted glyoxalylamides, steric effects lead to the occurrence of both rotamers, as observed by NMR spectroscopy. The predominant peak corresponds with the anti rotamer, in which the bulkier alkyl group is orientated trans to the amide carbonyl group. In monoalkylated glyoxalylamides, only one set of peaks is observed, consistent with the presence of only one rotamer. Crystal structures of 5-methoxyindole-3-yl-N-tert-butylglyoxalylamide, indole-3-yl-N-tert-butylglyoxalylamide, and indole-3-yl-N-isopropylglyoxalylamide reported here reveal a syn conformation held by an intramolecular N–H…O hydrogen bond.