Methylamine Dehydrogenase Research Articles

Cover Feature: Generation of Oxidoreductases with Dual Alcohol Dehydrogenase and Amine Dehydrogenase Activity (Chem. Eur. J. 10/2021)

Cover Feature: Generation of Oxidoreductases with Dual Alcohol Dehydrogenase and Amine Dehydrogenase Activity (Chem. Eur. J. 10/2021)

Economy Assessment for the Chiral Amine Production with Comparison of Reductive Amination and Transamination Routes by Multi-Enzyme System

Chiral amines are key building blocks for pharmaceuticals. Economic assessment of commercial potential of bioprocesses is needed for guiding research. Biosynthesis of (S)-α-methylbenzylamine (MBA) was selected as case study. For transamination route, transaminase coupled with glucose dehydrogenase and lactate dehydrogenase catalyzed the reaction with NADH (Nicotinamide adenine dinucleotide) regeneration. Amine dehydrogenase coupled with NADH oxidase, which catalyzed the reductive amination process. Comparison of biosynthesis cost by reductive amination and transamination routes was carried out. Economic assessment based on the framework of cost analysis and preliminary process information revealed that cost is greatly dependent on enzyme price. The results indicated that enhancing the activity of amine dehydrogenase by 4–5 folds can drop the unit price of reductive amination to $0.5–0.6/g, which make it competitive with transamination route.

Generation of Oxidoreductases with Dual Alcohol Dehydrogenase and Amine Dehydrogenase Activity.

The l‐lysine‐ϵ‐dehydrogenase (LysEDH) from Geobacillus stearothermophilus naturally catalyzes the oxidative deamination of the ϵ‐amino group of l‐lysine. We previously engineered this enzyme to create amine dehydrogenase (AmDH) variants that possess a new hydrophobic cavity in their active site such that aromatic ketones can bind and be converted into α‐chiral amines with excellent enantioselectivity. We also recently observed that LysEDH was capable of reducing aromatic aldehydes into primary alcohols. Herein, we harnessed the promiscuous alcohol dehydrogenase (ADH) activity of LysEDH to create new variants that exhibited enhanced catalytic activity for the reduction of substituted benzaldehydes and arylaliphatic aldehydes to primary alcohols. Notably, these novel engineered dehydrogenases also catalyzed the reductive amination of a variety of aldehydes and ketones with excellent enantioselectivity, thus exhibiting a dual AmDH/ADH activity. We envisioned that the catalytic bi‐functionality of these enzymes could be applied for the direct conversion of alcohols into amines. As a proof‐of‐principle, we performed an unprecedented one‐pot “hydrogen‐borrowing” cascade to convert benzyl alcohol to benzylamine using a single enzyme. Conducting the same biocatalytic cascade in the presence of cofactor recycling enzymes (i.e., NADH‐oxidase and formate dehydrogenase) increased the reaction yields. In summary, this work provides the first examples of enzymes showing “alcohol aminase” activity.

Purification of chimeric amine dehydrogenase using a tailor-made aqueous two-phase system - A case study

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.

UMI-linked consensus sequencing enables phylogenetic analysis of directed evolution

The success of protein evolution campaigns is strongly dependent on the sequence context in which mutations are introduced, stemming from pervasive non-additive interactions between a protein’s amino acids (‘intra-gene epistasis’). Our limited understanding of such epistasis hinders the correct prediction of the functional contributions and adaptive potential of mutations. Here we present a straightforward unique molecular identifier (UMI)-linked consensus sequencing workflow (UMIC-seq) that simplifies mapping of evolutionary trajectories based on full-length sequences. Attaching UMIs to gene variants allows accurate consensus generation for closely related genes with nanopore sequencing. We exemplify the utility of this approach by reconstructing the artificial phylogeny emerging in three rounds of directed evolution of an amine dehydrogenase biocatalyst via ultrahigh throughput droplet screening. Uniquely, we are able to identify lineages and their founding variant, as well as non-additive interactions between mutations within a full gene showing sign epistasis. Access to deep and accurate long reads will facilitate prediction of key beneficial mutations and adaptive potential based on in silico analysis of large sequence datasets.

Reductive Amination of Biobased Levulinic Acid to Unnatural Chiral γ-Amino Acid Using an Engineered Amine Dehydrogenase

Optically pure (S)-4-aminopentanoic acid is a pivotal precursor in the synthesis of therapeutic molecules and pyrrolidinone derivatives. Enantioselective reductive amination of levulinic acid catal...

NAD(P)H‐Dependent Enzymes for Reductive Amination: Active Site Description and Carbonyl‐Containing Compound Spectrum

AbstractThe biocatalytic asymmetric synthesis of amines from carbonyl compounds and amine precursors presents an important advance in sustainable synthetic chemistry. Oxidoreductases (ORs) that catalyze the NAD(P)H‐dependent reductive amination of carbonyl compounds directly to amines using amine donors present advantages complementary to those of amine transaminases (ATAs) with respect to selectivity, stability and substrate scope. Indeed some ORs accept alkyl and aryl amines as reaction partners enabling access to chiral secondary amine products that are not directly accessible using ATAs. Moreover, superior atom economy can usually be achieved as no sacrificial amines are required as with ATAs. In recent years a number of ORs that apparently catalyze both imine formation and imine reduction in the reductive amination of carbonyls has been identified using structure informed protein engineering, sequence analysis from natural biodiversity and increasingly a mixture of both. In this review we summarize the development of such enzymes from the engineering of amino acid dehydrogenases (AADHs) and opine dehydrogenases (OpDHs) to become amine dehydrogenases (AmDHs), which are active toward ketones devoid of any requisite carboxylate and/or amine functions, through to the discovery of native AmDHs and reductive aminases (RedAms), and the engineering of all of these scaffolds for improved or altered activity. Structural and mechanistic studies have revealed similarities, but also differences in the determinants of substrate binding and mechanism in the enzymes. The survey reveals that a complementary approach to enzyme discovery that utilizes both natural genetic resources and engineering can be combined to deliver biocatalysts that have significant potential for the industrial synthesis of chiral amines.magnified image

Continuous production of a chiral amine in a packed bed reactor with co-immobilized amine dehydrogenase and formate dehydrogenase

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.

Electronic State of the His/Tyr-Ligated Heme of BthA by Mössbauer and DFT Analysis.

The BthA protein from the microorganism Burkholderia thailandensis contains two hemes with axial His/OH2 and His/Tyr coordinations separated by the closest interheme distance of 14 Å. BthA has a similar structure and belongs to the same family of multiheme cytochrome c peroxidases as MauG, which performs long-range oxidation of the partner protein methylamine dehydrogenase. Magnetic Mössbauer spectroscopy of the diferric state of BthA corroborates previous structural work identifying a high-spin (His/OH2) peroxidatic heme and a low-spin (His/Tyr) electron transfer heme. Unlike MauG, addition of H2O2 fully converts the diferric form of BthA to a stable 2e- oxidized state, allowing a new assessment of this state. The peroxidatic heme is found to be oxidized to a canonical compound II, S = 1 oxoiron(IV) heme. In contrast, the electronic properties of the oxidized His/Tyr heme are puzzling. The isomer shift of the His/Tyr heme (0.17 mm/s) is close to that of the precursor S = 1/2 Fe3+ heme (0.21 mm/s) which suggests oxidation of the Tyr. However, the spin-dipolar hyperfine coupling constants are found here to be the same as those for the ferryl peroxidatic heme, indicating that the His/Tyr heme is also a compound II, S = 1 Fe4+ heme and ruling out oxidation of the Tyr. DFT calculations indicate that the unusually high isomer shift is not attributable to the rare axial His/Tyr heme coordination. The calculations are only compatible with spectroscopy for an unusually long Fe4+-OTyr distance, which is presumably under the influence of the protein environment of the His/Tyr heme moiety in the H2O2 oxidized state of the protein. The results offer new insights into how high valence intermediates can be tuned by the protein environment for performing long-range oxidation.

Protein-inorganic calcium-phosphate supraparticles as a robust platform for enzyme co-immobilization.

Immobilization of enzymes provides many benefits, including facile separation and recovery of enzymes from reaction mixtures, enhanced stability, and co-localization of multiple enzymes. Calcium-phosphate-protein supraparticles imbued with a leucine zipper binding domain (ZR ) serve as a modular immobilization platform for enzymes fused to the complementary leucine zipper domain (ZE ). The zippers provide high-affinity, specific binding, separating enzymatic activity from the binding event. Using fluorescent model proteins (mCherryZE and eGFPZE ), an amine dehydrogenase (AmDHZE ), and a formate dehydrogenase (FDHZE ), the efficacy of supraparticles as a biocatalytic solid support was assessed. Supraparticles demonstrated several benefits as an immobilization support, including predictable loading of multiple proteins, structural integrity in a panel of solvents, and the ability to elute and reload proteins without damaging the support. The dual-enzyme reaction successfully converted ketone to amine on supraparticles, highlighting the efficacy of this system.

Separate Sets of Mutations Enhance Activity and Substrate Scope of Amine Dehydrogenase

AbstractMutations 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.

Methylotenera oryzisoli sp. nov., a lanthanide-dependent methylotrophic bacteria isolated from rice field soil.

A new lanthanide (Ln3+)-dependent methanol-utilizing bacterial strain, La3113T, was isolated from rice field soil and its taxonomic position was investigated using polyphasic approaches. The strain was aerobic, Gram-stain-negative, strongly motile, catalase-positive and cytochrome oxidase-positive. It could neither catalyse the hydrolysis of urea nor reduce nitrate to nitrite. Growth was observed within a temperature range of 10-40 °C and a pH range of 6-8, with optimum growth at 28 °C and pH 7. Methylamine was utilized as the single source of energy, carbon and nitrogen, and it was oxidized by methylamine dehydrogenase. C16 : 1 ω7c, C16 : 1 ω6c and C16 : 0 were the dominant cellular fatty acids. Its draft genome (2.67 Mbp and 44.9 mol% G+C content) encodes genes including three Ln3+-dependent methanol dehydrogenase (XoxF-type MDH) genes, those for formaldehyde assimilation (ribulose monophosphate pathway), formate dehydrogenases and methylamine dehydrogenases, but not Ca2+-dependent MDH (MxaFI-MDH), which characterizes the species as a Ln3+-dependent methylotroph. The 16S rRNA gene sequence showed that strain La3113T belongs to the genus Methylotenera and is closely related to Methylotenera mobilis JLW8T (98.29 % identity). The digital DNA-DNA hybridization (dDDH) values (less than 30 %) and average nucleotide identity (ANI) values (less than 85 %) between genomes of strain La3113T and related type strains were lower than the thresholds for species delineation (70 % for dDDH and 95-96 % for ANI). On the basis of these polyphasic approaches, we propose a novel Methylotenera species, Methylotenera oryzisoli sp. nov. (type strain La3113T=NBRC 111954T=DSM 103219T).

Kinetic Resolution of Racemic Primary Amines Using Geobacillus stearothermophilus Amine Dehydrogenase Variant.

A NADH‐dependent engineered amine dehydrogenase from Geobacillus stearothermophilus (LE‐AmDH‐v1) was applied together with a NADH‐oxidase from Streptococcus mutans (NOx) for the kinetic resolution of pharmaceutically relevant racemic α‐chiral primary amines. The reaction conditions (e. g., pH, temperature, type of buffer) were optimised to yield S‐configured amines with up to >99 % ee.

Enantioselective Bioamination of Aromatic Alkanes Using Ammonia: A Multienzymatic Cascade Approach

AbstractChiral amines are common drug building blocks and important active pharmaceutical ingredients. Preparing these functionalized compounds from simple materials, such as alkanes, is of great interest. We recently developed an artificial bioamination cascade for the C−H amination of cyclic alkanes by combining P450 monooxygenase, alcohol dehydrogenase, and amine dehydrogenase. Herein, this system has been extended to the synthesis of chiral aromatic amines. In the first hydroxylation step, process optimization increased the conversion to 77 %. Two stereoselectively complementary alcohol dehydrogenases and an amine dehydrogenase were selected for the bioconversion of aromatic hydrocarbons to amines. The amination reaction was optimized with respect to cofactor addition and enzyme dosage. Isopropanol was added to decrease ketone intermediate accumulation in the amination step, which further enhanced the overall conversion. This cascade system converted a panel of hydrocarbon substrates into the corresponding amines with excellent optical purity (>99 % ee) and moderate conversion ratios (13–53 %).

Data mining of amine dehydrogenases for the synthesis of enantiopure amino alcohols

Five amine dehydrogenases (AmDHs) derived from amino acid dehydrogenases have been identified and evaluated for the stereoselective amination of α-/β-functionalized carbonyl compounds to synthesize chiral amino alcohols.

Development of an engineered thermostable amine dehydrogenase for the synthesis of structurally diverse chiral amines

Structurally diverse chiral amines and amino alcohols were synthesized using an engineered thermostable amine dehydrogenase, demonstrating its extensive synthesis potential.

Copper-binding energetics of amicyanin in different folding states.

Amicyanin is a type I copper protein that mediates electron transfer between methylamine dehydrogenase and cytochrome c-551i for energy production in Paracoccus denitrificans. Although the Met98 axial ligand of amicyanin has been shown to dictate metal selectivity and specificity during protein folding, the mechanism involved in copper-mediated amicyanin folding is unknown. Here, we kinetically and spectroscopically described reaction steps for incorporating copper into fully and less folded apo-amicyanin and established thermodynamic parameters for two amicyanin folding states. The rate constant for the incorporation of copper into fully folded apo-amicyanin at 25 °C was almost 1.5-fold lower than that for the initial phase of copper addition to the less folded apo-amicyanin. However, the rate constant was 10-fold higher than that of the second phase of copper addition to less folded apo-amicyanin at 25 °C. When overall binding energetic parameters (ΔH° and ΔS°) for the incorporation of copper into fully folded apo-amicyanin were measured by the van't Hoff method and isothermal titration calorimetry, the values were more positive than those determined for less folded apo-amicyanin. This indicates that during amicyanin biogenesis, copper rapidly binds to an unfolded apo-amicyanin active site, inducing protein folding and favorably influencing subsequent organization of copper ligands.

Enantioselective Synthesis of Chiral Vicinal Amino Alcohols Using Amine Dehydrogenases

Chiral vicinal amino alcohols are an important motif found in many biologically active molecules. In this study, biocatalytic reductive amination of α-hydroxy ketones with ammonia was investigated ...

An Ammonium-Formate-Driven Trienzymatic Cascade for ω-Transaminase-Catalyzed (R)-Selective Amination.

(R)-Amination mediated by (R)-specific ω-transaminases generally requires costly d-alanine in excess to obtain the desired chiral amines in high yield. Herein, a one-pot, trienzymatic cascade comprising an (R)-specific ω-transaminase, an amine dehydrogenase, and a formate dehydrogenase was developed for the economical and eco-friendly synthesis of (R)-chiral amines. Using inexpensive ammonium formate as the sole sacrificial agent, the established cascade system enabled efficient ω-transaminase-mediated (R)-amination of various ketones, with high conversions and excellent ee (>99%); water and CO2 were the only waste products.

Generation of amine dehydrogenases with increased catalytic performance and substrate scope from \u03b5-deaminating L-Lysine dehydrogenase

Amine dehydrogenases (AmDHs) catalyse the conversion of ketones into enantiomerically pure amines at the sole expense of ammonia and hydride source. Guided by structural information from computational models, we create AmDHs that can convert pharmaceutically relevant aromatic ketones with conversions up to quantitative and perfect chemical and optical purities. These AmDHs are created from an unconventional enzyme scaffold that apparently does not operate any asymmetric transformation in its natural reaction. Additionally, the best variant (LE-AmDH-v1) displays a unique substrate-dependent switch of enantioselectivity, affording S- or R-configured amine products with up to >99.9% enantiomeric excess. These findings are explained by in silico studies. LE-AmDH-v1 is highly thermostable (Tm of 69 °C), retains almost entirely its catalytic activity upon incubation up to 50 °C for several days, and operates preferentially at 50 °C and pH 9.0. This study also demonstrates that product inhibition can be a critical factor in AmDH-catalysed reductive amination.