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Nitroreductases (NRs) hold promise for converting nitroaromatics to aromatic amines. Nitroaromatic reduction rate increases with Hammett substituent constant for NRs from two different subgroups, confirming substrate identity as a key determinant of reactivity. Amine yields were low, but compounds yielding amines tend to have a large π system and electron withdrawing substituents. Therefore, we also assessed the prospects of varying the enzyme. Several different subgroups of NRs include members able to produce aromatic amines. Comparison of four NR subgroups shows that they provide contrasting substrate binding cavities with distinct constraints on substrate position relative to the flavin. The unique architecture of the NR dimer produces an enormous contact area which we propose provides the stabilization needed to offset the costs of insertion of the active sites between the monomers. Thus, we propose that the functional diversity included in the NR superfamily stems from the chemical versatility of the flavin cofactor in conjunction with a structure that permits tremendous active site variability. These complementary properties make NRs exceptionally promising enzymes for development for biocatalysis in prodrug activation and conversion of nitroaromatics to valuable aromatic amines. We provide a framework for identifying NRs and substrates with the greatest potential to advance., Competing Interests: The authors declare no conflict of interest. “The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results”.

A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Amine dehydrogenases (AmDHs) catalyze the enzymatic reduction of ketones to amines, serving as a suitable biocatalytic route for amine synthesis. A limited number of experimentally validated native AmDHs (nat‐AmDHs) have been reported recently, expanding the sequences with this function to complement the small set of engineered enzymes. Since researchers can now probe into the vast diversity of enzymes within niche environments by a metagenomics approach, a tandem metagenomic and bioinformatic approach is a powerful tool to identify new members of limited enzyme families to access new features in an iterative fashion. The previously untapped biocatalytic reservoirs of the ocean environment and human microbiome were screened for potential AmDHs using a hidden Markov model. Among the hundreds of hits, a subset of 18 enzymes was selected for further characterization and were confirmed to display AmDH activity. Additional analysis on six enzymes confirmed altered cofactor specificities and variation in substrate scopes, catalytic efficiencies, and active site residues compared to the reference nat‐AmDHs previously described. Particularly, MATOUAmDH2 from an eukaryotic organism demonstrated specific activity of 11.07 and 0.88 U mg−1 toward isobutyraldehyde and 1,2‐cyclohexadione respectively. Their abundance among the screened environments was also described. The protein sequence diversity of validated AmDHs reached by this metagenomics mining strategy highlights the success of such an approach. Metagenomically mined proteins, including eukaryotic ones, stand to increase the reach of biocatalysis towards enviromentally benign processes. [ABSTRACT FROM AUTHOR]

Mutations were introduced into the leucine amine dehydrogenase (L‐AmDH) derived from G. stearothermophilus leucine dehydrogenase (LeuDH) with the goals of increased activity and expanded substrate acceptance. A triple variant (L‐AmDH‐TV) including D32A, F101S, and C290V showed an average of 2.5‐fold higher activity toward aliphatic ketones and an 8.0 °C increase in melting temperature. L‐AmDH‐TV did not show significant changes in relative activity for different substrates. In contrast, L39A, L39G, A112G, and T133G in varied combinations added to L‐AmDH‐TV changed the shape of the substrate binding pocket. L‐AmDH‐TV was not active on ketones larger than 2‐hexanone. L39A and L39G enabled activity for straight‐chain ketones as large as 2‐decanone and in combination with A112G enabled activity toward longer branched ketones including 5‐methyl‐2‐octanone. [ABSTRACT FROM AUTHOR]

The article focuses on the development of (R)-transaminases by applying a sequence-based algorithm to exclude sequences leading to known (S)-amine and (R)- or (S)-amino acid specificity. It states that the strategy in the work include evaluation of the related enzyme amino acid sequences to find sequence pattern in existing (R)- and (S)-amino acid. It demonstrates that developing sequences already extant in nature provide a more targeted path to new specificity than directed evolution.

Highlights • Engineered amine dehydrogenase shows different mechanism from its wild-type parent. • Effect of viscosity on reaction rate reveals an important conformational change. • Kinetic rate law has been developed for leucine amine dehydrogenase. • Substrate binding for leucine amine dehydrogenase is not strictly ordered. • Measured substrate and product inhibitions will inform reaction engineering. Abstract Leucine amine dehydrogenase (L-AmDH) was engineered from the leucine dehydrogenase (LeuDH) from Bacillus stearothermophilus through the introduction of two key mutations to the substrate binding pocket, thus removing affinity for keto-acids in favor of methyl ketones and enabling the production of chiral amines, important intermediates for pharmaceuticals. The present work examines the effects of the mutations on the kinetic properties of these two enzymes. We conclude that L-AmDH exhibits a random order of association of ketone and cofactor in the reductive amination direction, compared to the ordered sequential mechanism of LeuDH. In the oxidative deamination direction, an ordered sequential mechanism was confirmed for LeuDH, while a more complicated situation was found for L-AmDH. Kinetic solvent viscosity effects revealed an isomerization of enzyme-substrate complexes for both LeuDH reactions, but not for L-AmDH. Inhibition experiments showed stronger product inhibition for L-AmDH than for LeuDH, an important consideration for large-scale synthesis. [ABSTRACT FROM AUTHOR]

• IMAC resin immobilization yielded 6.5 d half life and ≤443 g/(L∙d) productivity. • Overall reaction rate is limited by excess enzyme over cofactor but not mass transfer. • As [S] < [E], the general rate law applies but not standard Michaelis-Menten kinetics. • Conversion or volumetric productivity can be optimized but not both simultaneously. A continuous flow packed bed reactor system was developed for the asymmetric reductive amination of 5-methyl-2-hexanone with a chimeric amine dehydrogenase (AmDH) and the formate dehydrogenase (FDH) from Candida boidinii. Both enzymes were co-immobilized onto the Nuvia® IMAC resin from Bio-Rad which employs the well-established binding chemistry between 6xHis-tagged proteins and immobilized Ni2+. Binding was robust under reaction conditions and continuous operation in a packed-bed flow column for more than five days was achieved. At 40 °C, conversion of 20 mM 5-methyl-2-hexanone ranged between 19 and 48%, with volumetric productivities between 443 and 107 g/L·day. The apparent biocatalyst half-life was 159 h and the absence of both internal and external mass transfer limitations was confirmed. Conversion was limited by modest competitive product inhibition of (R)-amine on ketone and an excess of enzyme over cofactor concentration, which precludes the use of Michaelis-Menten kinetics and results in a rate decrease of 80% compared to conditions where [S] ≫ [E]. The relationship between flow rate, conversion, and productivity was examined, with important lessons for reaction engineering. The reported work represents the first stable application of AmDHs in a continuous flow reactor for the production of chiral amines. [ABSTRACT FROM AUTHOR]

Enzymes used as biocatalysts in evolving green processes have already reached industrial competitiveness to classical chemical catalysts. After production of these enzymes, aqueous two-phase extraction using an aqueous two-phase system (ATPS) has been shown a promising and sustainable alternative to common cost-intense chromatographic purification. However, state-of-the-art ATPS design is based on "trial-and-error" screening approaches leading to un-optimized ATPS. Within this work, we demonstrate the applicability of our method for ATPS design to an industrially relevant chimeric amine dehydrogenase (cFL1). The method includes thermodynamic modeling of ATPS phase composition and investigations on the conformational and colloidal cFL1 stability to rapidly select a tailor-made ATPS, offering optimal process and processing conditions, outclassing the previously selected ATPS (identified by random screening) by a factor of 4. All investigations and the selection of the sodium glutamate-polyethylene glycol 2000 ATPS were performed in less than 4 weeks, demonstrating proof of concept for a fast and sustainable ATPS selection in an early stage of downstream processing development. Unlabelled Image • Case study on rapid selection of a tailor-made aqueous two-phase system • Aqueous two-phase extraction for purification of an industrial enzyme • Hybrid approach for fast preselection of suitable phase-formers • Conformational and colloidal enzyme stability investigations • Switchable enzyme partitioning through addition of displacement agent [ABSTRACT FROM AUTHOR]