No abstract

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.

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 Fe(3+) in such a "safe" manner in vitro. However, we previously calculated that the most abundant InsP, inositol hexakisphosphate, cannot interact with Fe(3+) in the presence of cellular concentrations of Mg(2+). In this work, we study the metal complexation behaviour of inositol 1,2,3-trisphosphate [Ins(1,2,3)P(3)], 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)P(3) with Na(+), K(+), Mg(2+), Ca(2+), Cu(2+), Fe(2+) and Fe(3+). Our calculations indicate that Ins(1,2,3)P(3) can be expected to complex all available Fe(3+) 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 Fe(3+) from Ins(1,2,3)P(3) under cellular conditions, and hence labile iron detected using this probe may include iron bound to Ins(1,2,3)P(3). Therefore Ins(1,2,3)P(3) is the first viable proposal for a transit iron ligand.

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.

Menthol isomers are high-value monoterpenoid commodity chemicals, produced naturally by mint plants, Mentha spp. Alternative clean biosynthetic routes to these compounds are commercially attractive. Optimization strategies for biocatalytic terpenoid production are mainly focused on metabolic engineering of the biosynthesis pathway within an expression host. We circumvent this bottleneck by combining pathway assembly techniques with classical biocatalysis methods to engineer and optimize cell-free one-pot biotransformation systems and apply this strategy to the mint biosynthesis pathway. Our approach allows optimization of each pathway enzyme and avoidance of monoterpenoid toxicity issues to the host cell. We have developed a one-pot (bio)synthesis of (1R,2S,5R)-(-)-menthol and (1S,2S,5R)-(+)-neomenthol from pulegone, using recombinant Escherichia coli extracts containing the biosynthetic genes for an "ene"-reductase (NtDBR from Nicotiana tabacum) and two menthone dehydrogenases (MMR and MNMR from Mentha piperita). Our modular engineering strategy allowed each step to be optimized to improve the final production level. Moderate to highly pure menthol (79.1%) and neomenthol (89.9%) were obtained when E. coli strains coexpressed NtDBR with only MMR or MNMR, respectively. This one-pot biocatalytic method allows easier optimization of each enzymatic step and easier modular combination of reactions to ultimately generate libraries of pure compounds for use in high-throughput screening. It will be, therefore, a valuable addition to the arsenal of biocatalysis strategies, especially when applied for (semi)-toxic chemical compounds.