Letter pubs.acs.org/acscatalysis
Understanding and Overcoming the Limitations of Bacillus badius and Caldalkalibacillus thermarum Amine Dehydrogenases for Biocatalytic Reductive Amination Ahir Pushpanath, Elina Siirola, Amin Bornadel, David Woodlock, and Ursula Schell* Johnson Matthey Plc, 260 Cambridge Science Park, Cambridge CB4 0WE, United Kingdom S Supporting Information *
ABSTRACT: The direct asymmetric reductive amination of ketones using ammonia as the sole amino donor is a growing field of research in both chemocatalysis and biocatalysis. Recent research has focused on the enzyme engineering of amino acid dehydrogenases (to obtain amine dehydrogenases), and this technology promises to be a potentially exploitable route for chiral amine synthesis. However, the use of these enzymes in industrial biocatalysis has not yet been demonstrated with substrate loadings above 80 mM, because of the enzymes’ generally low turnover numbers (kcat < 0.1 s−1) and variable stability under reaction conditions. In this work, a newly engineered amine dehydrogenase from a phenylalanine dehydrogenase (PheDH) from Caldalkalibacillus thermarum was recruited and compared against an existing amine dehydrogenase (AmDH) from Bacillus badius for both kinetic and thermostability parameters, with the former exhibiting an increased thermostability (melting temperature, Tm) of 83.5 °C, compared to 56.5 °C for the latter. The recruited enzyme was further used in the reductive amination of up to 400 mM of phenoxy-2-propanone (c = 96%, ee (R) < 99%) in a biphasic reaction system utilizing a lyophilized whole-cell preparation. Finally, we performed computational docking simulations to rationalize the generally lower turnover numbers of AmDHs, compared to their PheDH counterparts. KEYWORDS: amine dehydrogenase, biocatalysis, reductive amination, chiral amine, asymmetric catalysis
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INTRODUCTION
shown to catalyze similar transformations, albeit with low conversions.5 The Bommarius group at Georgia Tech has engineered amine dehydrogenases (AmDHs) from naturally existing amino acid dehydrogenases (AADHs) through the introduction of two- to four-point mutations into selected scaffolds.6 These AmDHs have been shown to catalyze the reductive amination of a selection of prochiral ketones and have been used in elegant hydrogen borrowing dual-enzyme cascade reactions for the synthesis of chiral amines from alcohols.7 The initial engineering efforts by Bommarius et al. focused on two wild-type amino acid dehydrogenases: a phenylalanine dehydrogenase (PheDH) from Bacillus badius (Bbad-PheDH) and a leucine dehydrogenase from Bacillus stearothermophilus (Bstea-LeuDH). Since then, the applicability of the “two to four residue mutagenesis scheme” on different amino acid dehydrogenase scaffolds has been shown on a periodical basis, with engineered AmDHs from Rhodococcus sp. M4 and Exiguobacterium sibiricum representing the latest examples.7b,8 In addition, chimeric amine dehydrogenases have been constructed combining the Bbad-PheDH and Bstea-LeuDH.9
Direct asymmetric reductive amination of ketones is a highly desirable transformation for the synthesis of chiral amine intermediates, particularly for the pharmaceuticals industry. Both hydrogenation and transfer hydrogenation methods are available, catalyzed by Ru-, Ir-, and Rh-based catalysts.1 Of key note is the asymmetric reductive amination of functionalized ketones, such as β-keto esters and amides, in the presence of an ammonium salt, hydrogen, and Ru-catalyst which has found many applications, including the synthesis of sitagliptin.1a However, the organometal-catalyzed reactions have certain limitations, namely low stereoselectivities for the reactions of nonactivated ketones, and the formation of the alcohol side product, because of the direct reduction of the ketone. Furthermore, the reaction equilibrium between the amine and the ketone does not favor imine formation, especially if water is present in the system. As an alternative to organometallic catalysts, biocatalysts have been widely studied for (formal) reductive aminations.2 Applications directly utilizing ammonia as an amino source, however, have, until recently, been limited to amino acid dehydrogenase-catalyzed synthesis of amino acids from the corresponding keto acids3 or, indirectly, utilizing alanine dehydrogenase-based equilibrium shift in transaminase chemistry.4 Very recently, imine reductases have also been © 2017 American Chemical Society
Received: February 15, 2017 Revised: March 25, 2017 Published: March 30, 2017 3204
DOI: 10.1021/acscatal.7b00516 ACS Catal. 2017, 7, 3204−3209
Letter
ACS Catalysis Scheme 1. Reductive Amination of Selected Ketones 1a−1e Catalyzed by Amine Dehydrogenases in This Work
Table 1. Kinetic Parameters of Purified Bbad-AmDH and Cal-AmDH for Six Substratesa kcat (s−1) substrate 1a 1b 1c 1d 1e 1f NADHb NH3c
Bbad-AmDH 6.37 4.04 0.39 0.005 0.044 0.29 0.64 0.6
± ± ± ± ± ± ± ±
0.46 0.21 0.01 0.0005 0.005 0.04 0.03 0.02
KM (mM) Cal-AmDH 11.33 3.46 0.35 0.007 0.007 0.06 0.84 0.6
± ± ± ± ± ± ± ±
Bbad-AmDH
0.41 0.16 0.01 0.0015 0.001 0 0.05 0.02
7.72 5.65 4.09 7.4 21.25 72.15 36.09 526.7
± ± ± ± ± ± ± ±
1.16 0.69 0.51 1.39 3.29 11.34 7.29d 54.45
Cal-AmDH 8.12 4.34 2.22 9.71 2.59 10.57 86.42 838.6
± ± ± ± ± ± ± ±
0.61 0.54 0.19 3.33 0.93 1.06 13.63d 66.93
a The reactions were performed at 200 μL scale in 96-well microtiter plates at 25 °C and the decrease in absorbance at 340 nm, indicating NADH depletion, was monitored using a MultiSkan GO UV-spectrometer (Thermo Fisher Scientific). The reaction buffer contained 2 M ammonia/ ammonium formate at pH 9.6, 200 μM NADH with varying concentration of substrate. The initial rate and Michaelis Menten function fitting was performed using GraphPad Prism 6.07 software for Windows. bDetermined by varying [NADH] in the presence of fixed concentration, 20 mM 1c and 2 M NH3. cDetermined by varying [NH3] in the presence of fixed concentration of 200 μM NADH and 20 mM 1c. dKM units are given in μM.
Finally, we performed computational docking simulations to rationalize the generally lower turnover numbers of AmDHs, compared to their AADH counterparts.
A comparative exploration of the substrate scope of a selection of these enzymes has also been recently shown, highlighting their versatility.10 Despite the existence of this growing class of engineered enzymes, the scalability of the reactions they catalyze has not been demonstrated to industrially desirable standards (e.g., for pharmaceuticals, product concentrations of >50 g/L are required, with a biocatalyst yield of 10−100 gproduct/gimmobilized biocatalyst).11 The factors limiting the overall conversions achieved with biocatalysts generally include the stability of the enzyme under process conditions, substrate and product inhibitions, unfavorable equilibriums, and catalytic parameters.12 Wild-type amino acid dehydrogenases generally have turnover numbers (kcat, s−1) of ∼10013 and have been widely used in industrial applications.3 However, the fastest amine dehydrogenase reported to date from B. badius only has a kcat of 6.85 ± 0.59 s−1 for the preferred substrate 4fluorophenylacetone (1a, Scheme 1). Although this value should be sufficient assuming that a general threshold value for a useful biocatalyst is usually >1 s−1,12b the use of AmDHs in synthesis of chiral amines has still not been demonstrated past 80 mM for the same substrate.14 Here, we studied the kinetic parameters of the existing B. badius AmDH for favorable and unfavorable substrates, attempted to engineer an increase in activity for the latter substrates and observed susceptibility to mutation in the active site. This prompted a genome mining approach to recruit and mutate an amino acid dehydrogenase to yield a functional AmDH with a very similar kinetic profile to Bbad-AmDH but an increased melting temperature (Tm; defined as the temperature at which 50% of the protein is unfolded). Both enzymes were then applied as lyophilized lysate or lyophilized cells in the synthesis of 400 mM (R)-1phenoxypropan-2-amine (2c, Scheme 1).
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RESULTS AND DISCUSSION Bbad-AmDH has been shown to have elevated activity toward a handful of ketones, while the majority are being converted with specific activities in the low mU/mg range.6b In light of this, enzyme engineering of the Bbad-AmDH enzyme for improvement of the kinetic parameters for less-favored substrates was desirable. We performed a mutagenesis campaign focusing on a reduced alphabet (see Table S4 in the Supporting Information) and site saturation of four residues, delineating the active site pocket closest to the phenyl substituent of the docked substrate 1d (see Figure S3 in the Supporting Information). However, only a limited improvement of 1.9-fold specific activity increase for 1d was achieved (Table S7 in the Supporting Information). Determination of the melting points of the created variants showed that some variants had marked changes in thermostability, with even one mutation, V309L, showing a 12.5 °C drop in Tm (see Figure S9 and Table S13 in the Supporting Information). This was in agreement with previous data about mutation susceptibility in the active site of the same enzyme, where a drop of 4.4 °C was observed upon the introduction of the initial two mutations (K67S/N266L).6b Another amine dehydrogenase from E. sibiricum was generated with three mutations (K77S/N270I/V300C) and showed a Tm decrease of 10.7 °C, starkly reflecting the generally accepted tradeoff between the acquisition of new enzymatic functions and stability in enzymes (see Table S13).15 To this end, an amino acid dehydrogenase with higher natural thermostability was desirable to serve as a scaffold for engineering a stable AmDH. 3205
DOI: 10.1021/acscatal.7b00516 ACS Catal. 2017, 7, 3204−3209
Letter
ACS Catalysis Table 2. Specific Activities of Purified Bbad-AmDH and Cal-AmDH for a Panel of Substratesa
a The reactions were performed at 200 μL scale in 96-well microtiter plates at 25 °C, and the decrease in absorbance at 340 nm, indicating NADH depletion, was monitored using a MultiSkan GO UV-spectrometer (Thermo Fisher Scientific). The reaction buffer contained 2 M ammonia/ ammonium formate at pH 9.6, 200 μM NADH and
