Reshaping the Active Pocket of Amine Dehydrogenases for

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Reshaping the Active Pocket of Amine Dehydrogenases for Asymmetric Synthesis of Bulky Aliphatic Amines Feifei Chen, Gao-Wei Zheng, Lei Liu, Hao Li, Qi Chen, fulong li, Chun-Xiu Li, and Jian-He Xu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b04135 • Publication Date (Web): 15 Feb 2018 Downloaded from http://pubs.acs.org on February 18, 2018

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ACS Catalysis

Reshaping the Active Pocket of Amine Dehydrogenases for Asymmetric Synthesis of Bulky Aliphatic Amines Fei-Fei Chen†, Gao-Wei Zheng†, Lei Liu, Hao Li, Qi Chen, Fu-Long Li, Chun-Xiu Li, and Jian-He Xu* State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China ABSTRACT: The asymmetric reductive amination of ketones with ammonia using engineered amine dehydrogenases (AmDHs) is a particularly attractive and environmentally friendly method for the synthesis of chiral amines. However, one major challenge for these engineered AmDHs is their limited range of accepted substrates. Herein, several engineered AmDHs were developed through the evolution of naturally occurring leucine dehydrogenases, which displayed good amination activity toward aliphatic ketones but restricted catalytic scope covering short-chain substrates. Computational analysis helped identify two residues, located at the distal end of the substrate-binding cavity, that generate steric hindrance and prevent the binding of bulky aliphatic ketones. By fine-tuning these two key hotspots, the resulting AmDH mutants are able to accept previously inaccessible bulky substrates. More importantly, the mutations were also proved applicable for expanding the substrate scope of other homologous AmDHs with sequence identities as low as 70%, indicating a broad impact on the development of AmDHs and the synthesis of structurally diverse chiral amines. KEYWORDS: Amine dehydrogenase, reductive amination, chiral amine, protein engineering, biocatalysis, substrate scope

INTRODUCTION Chiral amines are important building blocks frequently used in the synthesis of numerous biologically active molecules in the pharmaceutical and agrochemical industries. According to a recent estimation, approximately 40% of active pharmaceutical ingredients (APIs) approved by the US Food and Drug Administration (FDA) in recent years contain one or more chiral amine moieties.1 During recent decades, considerable effort has been dedicated to the development of more efficient and sustainable chemical methods for the synthesis of chiral amines, as exemplified by the progress in classic asymmetric hydrogenation of imines/enamines and the emergence of some C–C and C–N bond formation methods. However, these chemical strategies suffer from many problems such as harsh reaction conditions, the use of precious and environmentallyunfriendly transition metal catalysts, poor stereoselectivity, and tedious amino protection and deprotection processes.2 With advances in protein engineering technologies, biocatalysis has emerged as a promising and environmentally benign alternative to traditional chemocatalysis for the synthesis of optically pure amines.3 Numerous biocatalytic routes catalysed by various enzymes including lipases,4 ωtransaminases,5 amine oxidases,6 lysases,7 imine reductases,8 reductive aminases,9 and amine dehydrogenases (AmDHs)10–17 have been successfully developed for the synthesis of chiral amines. Among them, AmDH-catalysed asymmetric reductive amination of prochiral ketones has attracted much interest as it can directly produce chiral amines using inexpensive ammonia as amino donor. Although a wild-type (WT) AmDH from Streptomyces virginiae with reductive amination activity has been reported, the stereoselectivity was poor and thus far the corresponding gene/protein sequence has not been disclosed.18 Recently, Bommarius and coworkers first reported the development of two engineered AmDHs after several rounds evolution of naturally occurring amino acid dehydrogenases

(AADHs),10 and the construction of a chimeric AmDH 11 by domain shuffling of the two engineered enzymes. These engineered AmDHs enable the direct synthesis of chiral amines from ketones (without any carboxyl group) using a coupled glucose dehydrogenase (GDH) or formate dehydrogenase (FDH) cofactor recycling system (Scheme 1).

Scheme 1. Amine Dehydrogenase-catalysed Asymmetric Reductive Amination of Ketones with Ammonia. Inspired by the engineering strategy of Bommarius and colleagues, ourselves and another group successfully developed a respective AmDH from Exiguobacterium sibiricum 12 and Rhodococcus sp. M4.13 By combining our engineered AmDH with a screened alcohol dehydrogenase (ADH) with poor enantioselectivity, we subsequently constructed a redox-neutral two-enzyme cascade for asymmetrically transforming racemic secondary alcohols into chiral amines.12 Notably, a similar concept, dual-enzyme hydrogen-borrowing cascade, was also proposed and more systematically investigated by Turner and coworkers.14 More recently, Mutti and coworkers demonstrated the synthetic potential of the engineered AmDHs by conducting a thorough investigation on their catalytic repertoire, optimal reaction conditions and stereoselectivity using a variety of ketones and aldehydes.15 The applicability of AmDH was further substantiated by a potent biphasic reaction system employing a more thermostable AmDH from Caldalkalibacillus

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thermarum with substrate loading up to 400 mM.16 In addition, different enzyme immobilisation techniques also proved to be applicable to AmDHs with facile operation, elevated catalytic performance and lower catalyst cost.17 However, to date, only a limited number of AmDHs have been developed through engineering of existing WT AADHs as scaffolds. Moreover, one major issue with the engineered AmDHs is their narrow substrate scope. Expanding the substrate scope of engineered AmDHs based on structural information is therefore a major aim of many researchers. In the present study, we aimed to probe new residues that may affect the binding of bulky aliphatic ketones with AmDHs, and subsequently tailor them to expand the substrate scope of this class of enzymes.

RESULTS AND DISCUSSION Development of AmDHs by Engineering the Existing AADHs. Using a group of naturally occurring leucine dehydrogenases (LeuDHs) available in our laboratory as starting templates,19 several functional AmDHs with measurable activity toward aliphatic ketones were developed by introducing the same mutations (K68S/N261L) reported by Bommarius and colleagues (Table S2).10a Among them, three amine dehydrogenases, namely EsAmDH from Exiguobacterium sibiricum,12 LfAmDH from Lysinibacillus fusiformis, and BspAmDH from Bacillus sphaericus, exhibited both considerable catalytic efficiency and impressive thermostability (Table 1). LfAmDH possessed the longest half-life (t1/2 = 36.5 h) at 60°C, and exhibited the highest reductive amination activity (up to 3 U/mg protein) toward aliphatic ketones containing four or five skeletal carbon atoms, but an extremely low activity toward bulky aliphatic ketones such as 2-hexanone and no activity at all for some substrates such as 2-heptanone and 2-octanone (Table S3). Considering the limited substrate scope of the engineered AmDHs, further engineering to create new AmDH mutants with broader specificity is highly desirable. Herein, LfAmDH was chosen as a representative enzyme for investigation of substrate scope. Expanding the Substrate Scope of Engineered AmDHs. To more rationally identify the residues affecting the binding of bulky aliphatic ketones in AmDHs, a homology model of

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Table 1. Kinetic Parameters and Half-lives of AmDHs Engineered from Naturally Occurring Leucine Dehydrogenases (LeuDHs)a enzyme

Km (mM)

kcat (s–1)

kcat/Km (mM–1 s–1) t1/2 (min)b

EsAmDH

9.2 ± 0.3c

0.84 ± 0.01c

0.091c

450

LfAmDH

30.6 ± 2.0

1.7 ± 0.04

0.057

2190

BspAmDH

32.4 ± 1.7

2.2 ± 0.04

0.069

540

a

For determining kinetic parameters, activity was measured in NH4Cl/NH4OH buffer (2 M, pH 9.5) containing 0.1 mM NADH and varied concentration of 2-pentanone at 30°C. bHalf-life (t1/2) was determined by incubating purified enzyme at 60°C and measuring the residual activity toward 2-pentanone over time. cData from reference.12

LfAmDH was built based on the crystal structure of a leucine dehydrogenase from Bacillus sphaericus (PDB: 1LEH), which shares 86% sequence identity with LfAmDH. By docking the substrate 2-heptanone into the active site of LfAmDH, we revealed that the substrate-binding cavity cannot accommodate relatively bulky aliphatic substrates (Figure 1A). Based on the binding mode shown in Figure 1A, we assumed that residue A113 located at the distal end of the substrate-binding cavity may generate steric hindrance that prevents the binding of bulky ketones. Consequently, we initially mutated A113 to the smaller glycine. As anticipated, the single site mutation (LfAmDH-M1, A113G) resulted in a remarkable expansion of the substrate specificity to include longer-chain aliphatic ketones (Table 2, entries 1 and 2). Compared with the starting LfAmDH, LfAmDH-M1 displayed a 13-fold increase in specific activity toward 2-hexanone, and the emergence of activity (425 mU/mg) against the longer 2-hepanone. To further confirm our assumption, a saturation mutagenesis library comprising all 20 natural amino acids at position 113 was constructed.20 The results of activity assays show that only the LfAmDH-M1 (A113G) mutant exhibited improved activity toward 2-hexanone, while other mutants displayed significant decreases in activity and activity was completely lost in some cases, suggesting the replacement of A113 with bulky amino acids is detrimental to the activity of LfAmDH (Figure S2).

Figure 1. Illustration of the substrate-binding cavities of LfAmDH (A) and its mutant LfAmDH-M3 (A113G/T134G) (B) with docked substrate 2-heptanone. Residues surrounding the substrate-binding pocket are shown in surface representation, and 2heptanone (2-Hep) is shown as ball-and-stick model. Residues 113 and 134 responsible for steric hindrance, are shown as blue sticks in the wild-type LfAmDH and red sticks in mutant LfAmDH-M3 (A113G/T134G), respectively. The two catalytic residues (K80 and D115) and the main two residues initially mutated to generate AmDH activity (L261 and S68) are shown as orange and green sticks, respectively.

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ACS Catalysis Inspired by this result, we speculated that other residues surrounding the substrate-binding pocket may also generate steric hindrance and thereby prevent binding of bulky ketones. Alanine-scanning mutagenesis of the region surrounding G113 site was performed to identify other potential ‘hotspots’ for further expanding the substrate specificity, and T112, E114, T134 and V294 were selected as potential ‘hotspots’ for Alanine mutagenesis (Figure S3). Mutagenesis of these residues and subsequent activity assays of the resulting mutants revealed that A113G/T134A (LfAmDH-M2) displayed a 3.3-fold further increase in specific activity (1400 mU/mg) toward 2heptanone compared to A113G (LfAmDH-M1) (Table 2, entries 2 and 6). Notably, residue Thr134 is also located at the distal end of the substrate-binding cavity (Figure 1A), and thus is also likely to be a key residue for expanding the substrate-accessible space of LfAmDH. Subsequently, we further mutated A134 to the smaller glycine, and the resultant LfAmDH-M3 (A113G/T134G) mutant displayed even higher activity than the LfAmDH-M2 (A113G/T134A) mutant (Table 2, entries 6 and 7). The two crucial mutations, A113G and T134G, resulted in an obvious enlargement in the substrate-binding cavity, and consequently, the larger 2-heptanone substrate can be well accommodated in a relatively extended conformation (Figure 1B). This observation can be further supported by measuring the increased volume of substrate-binding pocket, which revealed that the volume of LfAmDH-M3 is 43 Å3 larger than that of the starting enzyme LfAmDH (Supporting Information).21 These results demonstrate that spacegenerating mutations can create additional substrate-binding volume in the binding cavity of LfAmDH which is beneficial to reduce steric hindrance and expand the range of accepted substrates.

mM–1 s–1, and 0.247 mM–1 s–1 for LfAmDH-M1, LfAmDH-M2, and LfAmDH-M3, respectively). Moreover, the enhancement in kcat/KM is mainly attributed to an increase in kcat (Figure 2B and 2C). Given that there is sometimes a trade-off between high enzymatic activity and enzyme stability, we determined the melting points (Tm) of the engineered mutants by circular dichroism measurements (Figure S4). Excitingly, all three mutants displayed high thermostability (Tm = 67.9°C, 70.0°C, and 68.1°C for LfAmDH-M1, LfAmDH-M2, and LfAmDH-M3, respectively), similar to the starting LfAmDH (Tm = 68.5°C). The Tm value of LfAmDH-M2 was even 1.5°C higher than that of the starting LfAmDH. These results suggest that the acquisition of the new enzymatic activity did not have a detrimental effect on stability, which is beneficial for the practical application of these mutants in chiral amine synthesis.

Table 2. Specific Activity of LfAmDH and Its Mutants toward Aliphatic Ketones with Varied Chain Lengths specific activity (mU mg–1 protein)a entry

mutant 2-pentanone

2-hexanone

2-heptanone

1

LfAmDH

930 ± 13

110 ± 6

n.a.b

2

A113G

220 ± 3

1470 ± 80

430 ± 0.8

3

A113G/T112A

270 ± 1.1

920 ± 30

300 ± 3

4

A113G/E114A

57 ± 3

360 ± 8

170 ± 10

5

A113G/V294A

130± 2

770 ± 50

430 ± 40

6

A113G/T134A

340 ± 19

1560 ± 30

1400 ± 26

7

A113G/T134G

670 ± 38

2240 ± 160

2590 ± 160

a Activity was measured in NH4Cl/NH4OH buffer (2 M, pH 9.5) containing 0.1 mM NADH and 20 mM substrate at 30ºC. bn.a. = no measurable activity.

Kinetic Parameters and Thermostability. To comparatively evaluate catalytic efficiency, kinetic parameters of the starting LfAmDH and its mutants were determined using 2pentanone, 2-hexanone, and 2-heptanone (Figure 2). It is shown that all mutants (LfAmDH-M1, LfAmDH-M2, and LfAmDH-M3) exhibited a great improvement in catalytic efficiency (kcat/KM) toward 2-hexanone and 2-heptanone compared with the starting LfAmDH (Figure 2A). For 2-heptanone, LfAmDH showed no detectable activity, while its three mutants exhibited a progressive increase in kcat/KM with the enlargement of substrate-accessible space (0.038 mM–1 s–1, 0.184

Figure 2. Comparison of kinetic parameters for LfAmDH, LfAmDH-M1 (A113G), LfAmDH-M2 (A113G/T134A), and LfAmDH-M3 (A113G/T134G) toward 2-pentanone, 2hexanone, and 2-heptanone. (A) Comparison of kcat/KM. (B) Comparison of kcat. (C) Comparison of KM. Kinetic constants of LfAmDH toward 2-heptanone were not measured due to the very low activity. Exploration of Substrate Scope. We further examined the activities of these mutants against a panel of ketones with different carbon chain lengths, including linear alkyl ketones, branched alkyl ketones, and a representative alkene ketone (Figure 3). The starting LfAmDH displayed good activity toward aliphatic ketones 1 and 2 that have fewer than six carbon atoms in the skeleton, but very low or even no measureable activity toward bulky substrates. By contrast, its three mutants with enlarged substrate-binding pockets displayed much

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Figure 3. Reductive amination activity of LfAmDH and its mutants toward aliphatic ketones with different carbon chain lengths. Table 3. Reductive Amination of Aliphatic Ketones with Varied Carbon Chain Lengths by LfAmDH and its Mutantsa enzyme

conversion (%)b

ee (%) (Config.)b

LfAmDH

>99

>99 (R)

LfAmDH-M1

86

>99 (R)

3

LfAmDH-M2

97

>99 (R)

4

LfAmDH-M3

98

>99 (R)

5

LfAmDH

99

>99 (R)

LfAmDH-M1

>99

>99 (R)

7

LfAmDH-M2

>99

>99 (R)

8

LfAmDH-M3

>99

>99 (R)

9

LfAmDH

4

>99 (R)

LfAmDH-M1

99

>99 (R)

11

LfAmDH-M2

>99

>99 (R)

12

LfAmDH-M3

>99

>99 (R)

13

LfAmDH

0

n.d.c

LfAmDH-M1

45

>99 (R)

15

LfAmDH-M2

61

>99 (R)

16

LfAmDH-M3

80

>99 (R)

17

LfAmDH

0

n.d.c

LfAmDH-M1

0

n.d.c

19

LfAmDH-M2

12

>99 (R)

20

LfAmDH-M3

22

>99 (R)

entry

substrate

1 2 2-pentanone

6 2-hexanone

10 2-heptanone

14 2-octanone

18 2-nonanone

a

Reaction conditions: 1.5 mg AmDH (purified protein), 4 mg FDH (crude cellfree extract), 50 mM substrate, 1 mM NAD+, DMSO (2% v/v for 2-pentanone and 2-hexanone, 4% v/v for 2-heptanone, 2-octanone and 2-nonanone), NH4COOH/NH4OH buffer (1 M, pH 8.8), 1 mL total volume, 30ºC, 180 rpm for 24 h. bDetermined by GC analysis (Supporting Information). cn.d. = not detected due to low activity.

improved activity toward longer-chain aliphatic ketones. For LfAmDH-M3 with the largest substrate-binding cavity, a carbon chain length of up to 10 atoms was accepted, and activity toward substrates 6− −10 was observed, albeit at very low levels for 2-nonanone and 2-decanone (14 mU mg–1 and 2.5 mU mg–1, respectively). Analytic Scale Synthesis of Chiral Amines. The reactivity of LfAmDH and its three mutants was assayed using ketones with varied carbon-chain lengths as substrates (Table 3). The results show that the starting LfAmDH exhibited great reactivity toward short-chain aliphatic ketones such as 2-pentanone and 2-hexanone but only trace conversion toward 2-heptanone (entry 9) and even no measurable conversions toward longerchian aliphatic ketones (entries 13 and 17), while the mutants displayed not only excellent conversion toward short-chain ketones but also considerable or emerging conversions toward bulky aliphatic ketones. Among these mutants, LfAmDH-M3 afforded the highest conversions in the amination of bulky aliphatic ketones. Furthermore, all mutants maintained the enantioselectivity of the starting LfAmDH, and yield (R)configuration products with >99% ee, suggesting that the space-generating mutations in the binding cavity do not affect the specific orientation of the substrates in the pocket.22 To synthesize the enantiomeric amines with an opposite configuration, (S)-selective AmDHs need to be developed. We speculate that (S)-selective AmDHs might be accessible by either inverting the stereoselectivity of (R)-AmDHs or evolving the existing D-amino acid dehydrogenases through protein engineering techniques.23 Synthesis of (R)-2-Heptanamine on a 10 mL Scale. The reductive amination of 2-heptanone catalysed by LfAmDH and its mutants was scaled up to 10 mL to further evaluate the feasibility of the biocatalytic process (Figure 4). The three pocket-extended mutants continued to display pronounced activity toward 2-heptanone, while the starting LfAmDH exhibited extremely low reactivity and afforded only 97% conversion within 6 h and >99% conversion within 12 h, followed by LfAmDH-M2 that achieved >93% conversion within 6 h and >99% conversion within 12 h. LfAmDH-M1 also achieved almost full conver-

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ACS Catalysis sion (99%) within 24 h, albeit at a lower initial reaction rate than LfAmDH-M2 and LfAmDH-M3, indicating that all three mutants are robust under the reaction conditions. Moreover, the continual transformation of ketone to amine during the initial 12 h implies that these mutants possess considerable operational stability under the reaction conditions, which is in agreement with their high Tm values.

sequence identity with LfAmDH, respectively) to achieve the same results, suggesting their general applicability in expanding the catalytic scope of other homologous AmDH enzymes. Table 4. Specific Activity (mU mg–1 protein) of EsAmDH and BspAmDH Mutants toward Aliphatic Ketones with Varied Carbon Chain Lengthsa mutation

Figure 4. Time courses of reductive amination of 2-heptanone by LfAmDH and its mutants. Validation of the Identified Hotspots in Other AmDHs. To evaluate the general applicability of these two identified hotspots (A113G and T134G) for expanding the substrate scope, these mutations were engineered into the other two aforementioned AmDHs, EsAmDH from Exiguobacterium sibiricum and BspAmDH from Bacillus sphaericus, which share 70.1% and 95.6% amino acid sequence identity with LfAmDH, respectively. Satisfyingly, incorporation of these two space-generating mutations also enabled the acceptance of bulkier aliphatic ketones, and significantly enhanced activity toward them (Table 4). In accordance with the results of the LfAmDH mutants, the A113G/T134G (A122G/T143G in EsAmDH) mutation in BspAmDH resulted in the greatest increase in activity toward longer-chain aliphatic ketones, while mutation of A113G (A122G in EsAmDH) and A113G/T134A (A122G/T143A in EsAmDH) also markedly enhanced the enzyme activity. These results confirm that the identified hotspots can be manipulated in other AmDHs sharing adequate homology with LfAmDH to create new AmDH mutants with significantly elevated activity toward bulky aliphatic ketones. Therefore, this substrate-binding space generating mutagenesis approach might be an effective and universal approach for expanding the catalytic scope of homologous AmDHs in a minimalist fashion.

CONCLUSIONS We successfully identified two key residues surrounding the substrate-binding pocket in AmDHs that affect the binding of bulky aliphatic ketones, and subsequently developed several AmDH mutants with significantly increased substrate acceptance by tailoring these two key sites using the starting AmDH as template. The best mutant displayed significantly enhanced catalytic efficiency toward bulky aliphatic ketones with more than six atoms in the carbon skeleton such as 2heptanone and 2-octanone, without compromising the robust stability and excellent stereoselectivity (>99% ee) of the WT enzyme. More significantly, these key residues can be manipulated in other two AmDHs (sharing 70% and 96% amino acid

2-pentanone

2-hexanone

2-heptanone

EsAmDH

800 ± 30

83 ± 0.4

n.a.b

EsAmDH-A122G

220 ± 20

820 ± 40

350 ± 20

EsAmDH-A122G/T143A

240 ± 14

1000 ± 40

700 ± 60

EsAmDH-A122G/T143G

460 ± 25

1430± 60

1480 ± 25

BspAmDH

1030 ± 17

89 ± 1.1

n.a.b

BspAmDH-A113G

240 ± 20

1290 ± 20

370 ± 20

BspAmDH-A113G/T134A

380 ± 2

1340 ± 30

1350 ± 14

BspAmDH-A113G/T134G

750 ± 9

2360 ± 10

2800 ± 90

a

Activity was measured under the same conditions as described in Table 2. bn.a. = no measurable activity.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. Chemicals, mutagenesis, protein expression and purification, enzyme assay and screening, kinetic analysis, enzyme stability studies, protein homo-modeling and molecular docking, biotransformation, analytic method, GC chromatograms, NMR analysis.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

Author Contributions † The authors contributed equally to this work (F.-F. Chen and G.W. Zheng).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (Nos. 21472045 and 21536004).

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