Structure-Guided Steric Hindrance Engineering of Bacillus Badies Phenylalanine Dehydrogenase for Efficient L-Homophenylalanine Synthesis

doi:10.21203/rs.3.rs-692979/v1

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Structure-Guided Steric Hindrance Engineering of Bacillus Badies Phenylalanine Dehydrogenase for Efficient L-Homophenylalanine Synthesis

https://doi.org/10.21203/rs.3.rs-692979/v1

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Background: Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block L-homophenylalanine (L-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading.

Results: In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badies to create an enhanced biocatalyst for efficient L-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic activity. Seven superior mutants were acquired, and the optimal triple-site mutant V309G/L306V/V144G showed a 12.9-fold higher k_cat/K_m value than wild-type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to L-HPA in 210 min, and the specific space-time conversion was 30.9 mmoL·g⁻¹·L⁻¹·h⁻¹. The substrate loading and specific space-time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones.

Conclusions: Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of L-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

Biotechnology and Bioengineering

L-homophenylalanine

phenylalanine dehydrogenase

steric hindrance

enzyme engineering

catalytic efficiency

reductive amination

L-homophenylalanine (L-HPA) is an unnatural aromatic amino acid and contains a single-carbon extended side-chain compared with L-phenylalanine [1]. L-HPA can serve as a versatile chiral building block for the synthesis of several blockbuster pharmaceuticals, such as angiotensin-converting enzyme inhibitors [2, 3], proteasome inhibitors [4, 5], acetylcholinesterase inhibitors [6], and β-lactam antibiotics [6]. Chemical methods of L-HPA synthesis are constrained by lengthy steps, high cost, environmental pollution, and poor enantioselectivity, rendering industrial processes ultimately unsustainable [7, 8]. By contrast, biocatalysts are environmentally friendly, mild reaction conditions, and have excellent selectivity [9, 10]. Thus, various enzymatic routes including kinetic resolution catalyzed by lipase [11], in situ racemization catalyzed hydantoinase [12, 13], and asymmetric transamination catalyzed by transaminase [14, 15] were recently developed and applied to L-HPA synthesis. Nevertheless, the foregoing reactions also have limitations such as a theoretical maximum 50% lipase yield, poor applicability because of the strict D-enantioselectivity of hydantoinase, and undesirable equilibrium shifts in the reversible transaminase transamination [16].

Direct asymmetric reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is a promising approach in L-HPA synthesis. It consumes only free ammonia and generates only water as a byproduct (Fig. 1). Asano et al. [17] reported the use of PheDH from Bacillus sphaericus (BsPheDH) to prepare L-HPA. Bradshaw et al. [18] investigated the application of recombinant E. coli cells harboring PheDH from Rhodococcus sp. M4 (RsPheDH) to produce L-HPA. They achieved 63% conversion using 60 mM substrate. Ahmad et al. [19] enhanced the applicability of RsPheDH in L-HPA production via cell immobilization. They achieved > 80% conversion using 67 mM substrate. Notably, Zhang et al. [20] stated that the combination of substrate fed-batch with continuous product removal improved substrate loading in L-HPA production catalyzed by PheDH from Thermoactinomyces intermedius (TiPheDH). A final substrate concentration of 510 mM was achieved after eight cycles of a fed-batch operation. An important constraint in PheDH-catalyzed L-HPA production is the poor catalytic efficiency toward non-native, bulky 2-OPBA compared with native phenylpyruvic acid (PPA). Consequently, the substrate loading is low and fails to meet the requirements of industrially and commercially viable biocatalysts and biocatalysis processes [21].

Directed evolution techniques are powerful tools for constituting a prolific source of biocatalysts [22, 23]. Steric hindrance of the substrate-binding pocket plays a significant role in modulating the catalytic properties of an enzyme, including enantioselectivities [24, 25], substrate specificities [26, 27], and catalytic activities [25, 28, 29]. Recent enzyme engineering studies focused on increasing or conferring enzyme activity towards non-native bulky substrates by attenuating the steric hindrance of the substrate-binding pocket [30, 31]. The transaminase ATA-117 mutant constructed by Merck and Codexis [32] is a remarkable example of such engineering, as the mutant showed marginal reductive amination activity toward the bulky prositagliptin ketone, which was successfully used to catalyze the synthesis of the antidiabetic compound sitagliptin. Chen et al. [33] used rational design based on steric hindrance attenuation to induce asymmetric reductive amination activity of engineered amine dehydrogenases towards several previously unreactive long-chain α-aliphatic ketones. Zheng et al. [34] engineered hydroxynitrile lyase mutants remarkably increased the asymmetric hydrocyanation activity toward the rigid pharmaco aldehydes, and successfully applied them to achieve the synthesis of the β₂-adrenoreceptor agonist salmeterol. These successful advances demonstrated the feasibility of steric hindrance engineering of substrate-binding pockets to modulate the catalytic activity of enzymes towards non-native, bulky substrates.

In the present study, we report structure-guided steric hindrance engineering of PheDH from Bacillus badies (BbPheDH) to improve its catalytic efficiency in L-HPA synthesis. Steric hindrance of the substrate-binding pocket of BbPheDH was subjected to crucial residue identification and evaluation of the mutagenesis libraries constructed based on degenerate codons. Seven superior mutants were generated after four rounds of steric hindrance engineering. The optimal triple-site mutants significantly enhanced catalytic efficiency, substrate loading, and specific space-time conversion. Structure analysis elucidated the factors contributing to the enhanced catalytic efficiency observed in the enzyme kinetics and transformation experiments.

Preliminary activity studies and molecular modeling

Initially, two PheDHs from B. badies (BbPheDH) and G. kaustophilus (GkPheDH) were selected as candidate enzymes in the development of an enhanced biocatalyst for L-HPA synthesis. As 2-OPBA had a larger side-chain than the native substrate PPA, its catalysis by BbPheDH and GkPheDH was characterized by lower turnover frequencies (k_cat) and less favorable catalytic efficiency (k_cat/K_m) (Table 1), which motivated us to consider enhancing catalytic efficiency toward 2-OPBA by the steric hindrance engineering. Biotransformation experiments were subsequently performed using BbPheDH and GkPheDH as biocatalysis, giving 67.5% and 53.6% conversions at 0.2 M substrate concentration within 8 h (Table 1 and Additional file 2: Figure S1). Therefore, BbPheDH was selected as the starting mutagenesis template for steric hindrance engineering given the relatively high conversion.

Table 1 Kinetic parameters and conversions^a of BbPheDH and GkPheDH in the reductive amination of PPA and 2-OPBA

Enzyme	Substrate	k_cat (s^-1)	K_m (mM)	k_cat/K_m (mM^-1·s^-1)	Conversion (%)
BbPheDH	PPA	114.6 ± 5.6	0.31 ± 0.04	369.7	99.9^b
BbPheDH	2-OPBA	10.7 ± 0.8	0.63 ± 0.06	17.0	67.5^c
GkPheDH	PPA	91.8 ± 5.6	0.27 ± 0.04	340.0	99.9^b
GkPheDH	2-OPBA	5.8 ± 0.7	0.52 ± 0.04	11.2	53.6^c

^aReaction conditions: 0.1 M (0.2 M) 2-OPBA, 0.12 M (0.24 M) glucose, 0.5 mM NAD⁺, 1 M NH₄OH/HCOONH₄ buffer (pH 9.0), 10 g·L⁻¹ cell-free extracts of PheDH, and 12 g·L^-1 cell-free extracts of GluDH in a total volume of 5 mL at 30 °C and 200 rpm.

^bConversion was calculated with 0.1 M 2-OPBA.

^cConversion was calculated with 0.2 M 2-OPBA.

To assess the feasibility of developing an enhanced biocatalyst for efficient L-HPA synthesis, we first generated a structural homology model of BbPheDH (Figure 2A) based on the reported crystal structure of Rhodococcus sp. M4 PheDH [35] to develop hypotheses for mutagenesis library designs. The model revealed that the residues surrounding the catalytic active center of BbPheDH can be classified into two categories on the basis of their function in the substrate-binding process. Group one encompasses the catalytic residues that are appointed in the binding of the α-carboxylic and α-amino groups of the substrate, namely K78, K90, D125, and N276, respectively, which were highly conserved among the PheDH family (Additional file 2: Figure S2). These residues create a hydrophilic environment that combines the hydrophilic α-amino acid moiety of the substrate (Figure 2B). On the opposite side, a panel of hydrophobic residues creates a hindered hydrophobic environment forcing the substrate side-chain into its ideal binding pose (Figure 2C). Furthermore, limited structure-guided engineering was conducted on PheDHs and showed that most of the beneficial mutations introduced into the substrate-binding pocket were hydrophobic amino acid residues [36-38]. Based on the foregoing information, we selected amino acids with strong hydrophobicity and weak steric hindrance as target substitutes in the subsequent mutagenesis experiments.

Mutagenesis library construction and evaluation based on attenuated steric hindrance

To identify mutagenesis candidates for initial library construction, 2-OPBA was docked into the catalytic active center of BbPheDH to investigate the unfavorable hindrance interactions. Based on the acquired binding pose, all amino acid residues except the smallest glycine and the conserved catalytic residues within 6 Å of the side-chain phenyl ring of 2-OPBA were selected for site-directed mutagenesis in the first round of steric hindrance engineering (Figure 3A). Twenty-three single-site mutants were constructed at nine amino acid sites based on the designed degenerate codon (Additional file 1: Table S1). The initial activity assays of the cell-free extracts screened the three superior mutants L50V (M1-1), V309A (M1-2), and V309G (M1-3) (Additional file 2: Figure S3). Subsequent enzyme kinetics experiments demonstrated that the k_cat/K_m of these mutants towards 2-OPBA were 50.6 S^-1·mM^-1, 58.2 S^-1·mM^-1, and 73.8 S^-1·mM^-1, respectively, which were 3.0-fold, 3.4-fold, and 4.3-fold higher than wild-type (17.3 S^-1·mM^-1) (Figure 3F).

As mutant M1-3 had the highest catalytic efficiency towards 2-OPBA, it was selected as the mutagenesis template in the second round of steric hindrance engineering to investigate the potential synergistic effects. The binding pose of M1-3 with 2-OPBA indicated eight amino acid residues within 6 Å of the side-chain phenyl ring of 2-OPBA. For the rapid detection of any synergy of a proximal steric hindrance with residue G309, the residues were simplified by double-proximity filtering. We identified four mutagenesis candidates within 6 Å of both the phenyl ring of 2-OPBA and G309 (Figure 3B). Thus, a mutagenesis library consisting of ten double-site mutants was constructed, and the superior mutants V309G/L50V (M2-1) and V309G/L306V (M2-2) were screened by the initial activity assays (Additional file 2: Figure S4). The k_cat/K_m value of the mutants M2-1 and M2-2 toward 2-OPBA were determined to be 143.3 S^-1·mM^-1 and 178.8 S^-1·mM^-1, respectively, which were 8.4-fold and 10.5-fold higher than wild-type (Figure 3F).

With this promising result, the mutant M2-2 was selected as the template in the third round of steric hindrance engineering. Here, we investigated proximal steric hindrance synergy with V306. Three mutagenesis candidates were identified by double-proximity filtering using V306 as the key residue (Figure 3C). The initial activity assay of the ten triple-site mutants revealed two superior mutants V309G/L306V/V144A (M3-1) and V309G/L306V/V144G (M3-2) (Additional file 2: Figure S5). The k_cat/K_m value of mutants M3-1 and M3-2 toward 2-OPBA reached 192.5 S^-1·mM^-1 and 219.2 S^-1·mM^-1, respectively, which was 11.3-fold and 12.9-fold higher than that of wild-type (Figure 3F).

Inspired by the above results, we explored the possibility of increasing catalytic activity towards 2-OPBA using M3-2 as the template in the fourth round of steric hindrance engineering. Two mutagenesis candidates were identified (Figure 3D) and four corresponding quadruple-site mutants were constructed. However, none of them exhibited higher activity towards 2-OPBA than M3-2 (Additional file 2: Figure S6).

Together, seven superior mutants were successfully screened from the 47 elements in the site-directed mutagenesis library (Figure 3E). The optimal triple-sites mutant M3-2 (V309G/L306V/V144G) showed a 12.9-fold enhance in k_cat/K_m value compared with wild-type BbPheDH. Unlike the changes in binding affinity (K_m) (Figure 3H), the observed enhancements in catalytic efficiency (k_cat/K_m) (Figure 3F) are attributed mainly to the increases in turnover frequency (k_cat) (Figure 3G). Moreover, the thermostabilities of BbPheDH and its superior mutants were assessed by measuring the T₅₀³⁰ value. It is not unexpected that some kind of stability-activity tradeoff occurred in all mutants, although this tradeoff is of no practical importance since the T₅₀³⁰ value of all mutants still exceeded 50 ℃ (Additional file 1: Table S2).

Biocatalytic optimization analysis on the best mutant M3-2

The dependence of temperature and pH value were investigated for the reductive amination of 2-OPBA catalyzed by M3-2 paired with glucose dehydrogenase (GluDH) at 0.2 M substrate concentration. Figure 4A shows that 2-OPBA conversion was > 99% after 180 min when the reactions proceeded at 30–40 °C. Catalytic efficiency was slightly decreased at 25 °C because the reaction required 240 min to attain 99% conversion. At 50 °C, however, quantitative conversion was < 90% after 240 min. We then evaluated the effects of NH₄OH/HCOONH₄ buffer (1 M) at various pH values and 30 °C. Figure 4B shows that the fastest conversions occurred at pH 8.5 and the quantitative conversion was > 99% within 90 min. We subsequently performed the reductive amination of 2-OPBA in various concentrations of NH₄OH/HCOONH₄ buffer (pH 8.5) and NAD⁺ to determine the influences of NH₄⁺ and NAD⁺concentration. The 2-OPBA conversion was > 99% at 1–3 M NH₄⁺. By contrast, 4 M and 5 M NH₄⁺ lowered the conversion rate (Figure 4C). An activity assay indicated that low GluDH activity might explain the observed decrease in conversion rate in the presence of high NH₄⁺ concentrations (Additional file 2: Figure S7). Moreover, 2-OPBA can be fully converted whether the NAD⁺ concentration is 0.3 mM, 0.5 mM, or 1 mM. However, the conversion rate is < 50% when the NAD⁺ concentration is < 0.3 mM (Figure 4D).

Preparative scale synthesis of L-HPA employing mutant M3-2

Using the optimized reaction system (30 °C; pH 8.5; 3 M NH₄⁺; 0.3 mM NAD⁺), the preparative scale synthesis of L-HPA by reductive amination of 2-OPBA was carried out in 100 mL reaction volume. The pH value was maintained at 8.5 by titrating concentrated NH₃·H₂O during the reaction course. Figure 4E shows that 0.2 M and 0.3 M 2-OPBA were converted by > 99% conversion within 20 min and 30 min, respectively. However, substrate inhibition was observed at high concentrations. The quantitative conversion rates were < 70% and < 30% after 240 min in the presence of 0.4 M and 0.5 M substrate, respectively (Additional file 1: Table S3). This phenomenon was similar to that which was reported for TiPheDH from T. intermedius [39]. We speculate that the high substrate concentration may have an irreversible adverse effect on the enzyme [40].

Continuous substrate fed-batch L-HPA synthesis

Substrate concentration is a crucial factor in industrially feasible biocatalytic processes. Therefore, a combination of substrate fed-batch with product removal strategy was applied for continuous L-HPA synthesis. During the reaction course, 2-OPBA was repeatedly added to the initial NH₄OH/HCOONH₄ reaction medium (pH 8.5) containing 0.3 mM NAD⁺, 3 M NH₄⁺, 10 g L⁻¹ cell-free extracts of M3-2, and 12 g L⁻¹ cell-free extracts of GluDH. The substrate concentration was fixed at ~0.3 M to maintain high reaction efficiency. Meanwhile, L-HPA product was continuously precipitated from the reaction solution and isolated after filtration and drying. Consequently, 1.08 M 2-OPBA with a quantitative conversion of 90.2% was effectively transformed after four times fed-batch over 210 min (Figure 4F), and the specific space-time conversion reached 30.9 mmoL·g⁻¹·L⁻¹·h⁻¹. Comparisons with previously reported biocatalysis processes revealed that our M3-2 had the highest substrate loading and specific space-time conversion in L-HPA production (Additional file 1: Table S4). Furthermore, M3-2 displayed perfect stereoselectivity and yielded L-configuration HPA with up to 99% enantiomeric excess (ee). These results underscore the strong potential of M3-2 as a biocatalyst for continuous L-HPA synthesis.

Docking simulation and tunnel analysis of wild-type and derived mutants

To better understand the enhance in catalytic efficiency observed in enzyme-kinetics and biotransformation experiments at the atomic and molecular levels, we performed the docking simulations using the available structural information. The 2-OPBA was docked into the catalytic active center of the wild-type BbPheDH and the derived mutants M1-3, M2-2, and M3-2, respectively (Figure 5). The docking simulations revealed poor adaptability of 2-OPBA to the substrate-binding pocket of wild-type BbPheDH compared with the binding pose of the native substrate PPA (Additional file 2: Figure S8). The relatively bulky 2-OPBA side-chain could not fit into the substrate-binding pocket of wild-type BbPheDH in a relatively stretched configuration (Figure 5A and 5B), which may prevent the substrate from binding in the proper orientation for hydride transfer. By contrast, the incorporation of three crucial mutations V309G, L306V, and V144G gradually enlarged the volume of the substrate-binding pocket compared with that of BbPheDH. Hence, the relatively bulky phenyl group side-chain of 2-OPBA could fit in the enlarged substrate-binding pocket with a relatively stretched configuration at a high degree of freedom (Figure 5C-5H). Moreover, the observed enlargement of the substrate-binding pocket can be further supported by calculating the volume of the substrate-binding pocket, which revealed that the volume of the eventual mutant M3-2 was about 196.9 Å³ larger than that of the wild-type BbPheDH(Additional file 1: Table S5). These results demonstrate that the enlargement of the substrate-binding pocket in a reasonable range was beneficial for the interaction between the enzymes and the bulky substrates [31, 33].

Tunnels represent potential transport pathways for small molecules, water molecules, and ions, and play a significant role in the functioning of a large variety of proteins [41, 42]. The accessibility of a tunnel depends largely on its shape, size, and amino acid composition. To a certain extent, it can be modified by protein engineering [43, 44]. Therefore, we investigated whether steric hindrance engineering alters BbPheDH tunnels. Tunnels analyses based on the structure homology model of M3-2 revealed that compared with BbPheDH, the incorporation of V144G, L306V, and V309G formed a new tunnel (magenta) in the vicinity of the catalytic active center (Figure 6), suggesting a potential new transport pathway for substrate and product. This assumption was supported by the significantly increased turnover frequencies (k_cat) of 2-OPBA catalyzed by M3-2. Furthermore, V144G, L306V, and V309G mutations markedly altered the shapes of the existing tunnels (Figure 6). The widened tunnels (shown as red and orange) could accelerate the processes of substrates access and product egress. This observation demonstrated that the formation of new tunnels and widening of existing ones can modulate the catalytic activity of enzymes. It also resembled the findings reported for prior studies [45, 46].

Here, we employed a modified steric hindrance engineering approach to create an enhanced biocatalyst with BbPheDH as a mutagenesis template for efficient L-HPA synthesis. Seven superior mutants were screened from the mutagenesis libraries constructed based on molecular docking, double-proximity filtering, and a degenerate codon. The k_cat/K_m of mutant M3-2 (V309G/L306V/V144G) was 12.9-fold higher than that of wild-type, it converted 1.08 M 2-OPBA to L-HPA in 210 min, and its specific space-time conversion was 30.9 mmoL·g^− 1·L^− 1·h^− 1. The substrate loading and specific space-time conversion are the highest values reported so far, demonstrating that mutant M3-2 is a useful biocatalyst with potential for the industrial production of L-HPA. Structure analysis demonstrated that mutations altered the substrate-binding pocket and tunnel, and variations in steric hindrance modulate the catalytic activity of BbPheDH towards 2-OPBA. Moreover, our study indicates the combination of molecular docking with a double-proximity filter as a promising workflow for capturing the proximal steric hindrance synergistic effects. The modified steric hindrance engineering approach can be a valuable addition and extension of the current enzyme engineering toolbox.

Strains, plasmids, and chemicals

PheDHs from Bacillus badies (BbPheDH) and Geobacillus kaustophilus (GkPheDH) were generated in the laboratory. Glucose dehydrogenase from Bacillus amyloliquefaciens (BaGluDH) was purchased from Sigma-Aldrich Corp. (Beijing, China). Escherichia coli BL21 (DE3) and plasmid pET-28a (+) were procured from Novagen (Nanjing, China) and used as the gene expression host and vector, respectively. The 2-oxo-4-phenylbutyric acid (2-OPBA) and L-homophenylalanine (L-HPA) were acquired from J&K Chemical (Beijing, China). Isopropyl-β-D-thiogalactopyranoside (IPTG), kanamycin, NADH, and NAD⁺ were obtained from TCI (Shanghai, China). All other chemicals were purchased from commercial sources, at least reagent grade, and used without further purification.

Sequence alignment and site-directed mutagenesis

Amino acid sequence alignment was performed using the MUSCLE server (https://www.ebi.ac.uk/Tools/msa/muscle/) [47] and displayed using Esprit server (http://espript.ibcp.fr) [48]. Site-directed mutagenesis was induced by the whole-plasmid PCR approach using primers containing degenerate codons for mutagenesis, pET-28a (+) plasmid inserts, and the pdh template. The PCR products were analyzed by agarose gel electrophoresis and transformed into E. coli BL21 (DE3) cells. DNA sequencing was confirmed by Talen-Bio (Wuxi, China).

Protein expression and purification

Escherichia coli BL21 (DE3) cells containing recombinant pET-28a (+) plasmids were cultured in Luria-Bertani (LB) medium with kanamycin (50 mg·L^-1) at 37 °C and with shaking at 200 rpm for 2 h. Enzyme expression was induced with IPTG (final concentration = 0.2 mM) when the bacteria reached OD₆₀₀ = 0.6–0.8. The cells were incubated at 20 °C and with shaking at 200 rpm for 12 h. His-tagged enzymes were purified with an ÄKTA purifier system (GE Healthcare, Little Chalfont, UK). The cells were resuspended in buffer A (100 mM potassium phosphate, 150 mM NaCl, and 20 mM imidazole; pH 7.5) and disrupted with an ultrasonic cell disruption system (SCIENTZ-IID; Ningbo Scientz Biotechnology, Ningbo, Zhejiang, China) followed by centrifugation at 12,000 rpm and 4 °C for 30 min to remove cell debris and obtain cell-free extracts. The latter were passed through a 0.22-μm filter and loaded into a standard Ni-NTA affinity column (Thermo Fisher Scientific, Waltham, MA, USA) pre-equilibrated with buffer A. The column was then gradient-eluted with buffer B (100 mM potassium phosphate, 150 mM NaCl, and 500 mM imidazole; pH 7.5). Eluted fraction purity was determined by SDS-PAGE (Additional file 2: Figure S9).

Activity and kinetic parameters analysis

Reductive amination activity was determined for 200-μL aliquots in 96-well microtiter plates at 30 °C. The rate of initial decrease of the absorbance was measured at 340 nm in a MultiSkan GO UV-spectrometer (Thermo Fisher Scientific). The reaction mixture consisted of NH₄Cl/NH₄OH buffer (2 M; pH 9.5), 10 mM 2-OPBA, 0.2 mM NADH, and a certain amount of cell-free extract or purified enzyme. One unit of enzyme activity (1 U) was defined as the amount of enzyme oxidizing 1 μM NADH per minute. Kinetic parameters with respect to 2-OPBA were evaluated in NH₄Cl/NH₄OH buffer (2 M; pH 9.5) at 30 °C in the presence of 5–20 mM 2-OPBA and 0.5 mM NADH. Kinetic parameters with respect to NADH were evaluated in NH₄Cl/NH₄OH buffer (2 M; pH 9.5) at 30 °C in the presence of 0.1–0.5 mM NADH and 20 mM 2-OPBA (Additional file 1: Table S6). K_m and V_max were calculated in GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA) by nonlinear curve fitting of the initial rate vs. substrate concentration data to the Michaelis-Menten equation.

Thermostability analysis

Thermostability assays of wild-type BbPheDH and its mutant were conducted by measuring T₅₀³⁰, which is the temperature at which 50% of the enzyme activity is lost after 30 min heat treatment. The enzymes were incubated at various temperatures (30–70 °C) for 30 min, and their residual activity was measured. Specific activity before incubation was normalized as 100%. Enzyme activity was measured in NH₄Cl/NH₄OH buffer (2 M; pH 9.5) containing 0.2 mM NADH and 10 mM 2-OPBA.

Asymmetric reductive amination of 2-OPBA to L-HPA

PheDH was coupled with GluDH for the asymmetric reductive amination of 2-OPBA. The total volume of the biocatalytic reaction mixture was 5 mL. It contained cell-free extracts of E. coli wet cells (10 g·L^-1 PheDH and 12 g·L^-1 GluDH), 2-OPBA (0.1–0.5 M), glucose (0.12–0.6 M), NAD⁺ (0.05–1.0 mM), and NH₄OH/HCOONH₄ buffer (1–5 M; pH 7.5-9.5). The reaction mixture was incubated at 25–50 ºC with shaking at 200 rpm. Samples (200 μL) were drawn at intervals and alkalized with 200 μL NaOH (10 M) to terminate the reaction. The solutions were then passed through a 0.22-μm filter and subjected to High Performance Liquid Chromatography (HPLC).

Fed-batch synthesis of L-HPA

The initial reaction mixture (100 mL) consisted of cell-free extract (10 g·L^-1 PheDH and 12 g·L^-1 GluDH), 2-OPBA (0.3 M), glucose (0.36 M), NAD⁺ (0.3 mM), and NH₄OH/HCOONH₄ buffer (3 M; pH 8.5). The reaction was initiated by adding PheDH and GluDH and incubated at 30 °C with shaking at 200 rpm. The pH was maintained at 8.5 by titrating concentrated NH₃·H₂O during the reaction. Over the 30–75 min reaction period, the precipitated L-HPA was separated by filtration. The filtrate contained enzymes and cofactors and was used as the reaction medium for the next reaction period. The latter was initiated by adding 0.3 M 2-OPBA and 0.36 M glucose. Four fed-batch cycles were performed, and 2-OPBA conversion and the L-HPA enantiomeric excess (ee) value were determined by HPLC.

HPLC analysis

The conversion of 2-OPBA and the ee value of L-HPA were determined with an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA, USA). The absolute L-HPA configuration was established by comparing it with authentic reference material after derivatization. The conditions for determining the 2-OPBA conversion rate were as follows: Diamonsil C18(2) column (5 μm; 250 mm × 4.6 mm) (Phenomenex, Torrance, CA, USA); injection volume, 10 μL; mobile phase A (55% (v/v) methanol plus 0.1% (v/v) trifluoroacetic acid), 20 min at 30 °C; flow rate, 0.8 mL·min⁻¹; and UV detection at 230 nm. The 2-OPBA retention time was 9.04 min. The ee value of L-HPA was determined using a Develosil^® ODS-UG-5 column (5 μm; 150 mm × 4.6 mm) (Phenomenex) after derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide (FDAA). A 5-μL reaction sample, 4 μL of 1 M NaHCO₃, and 20 μL of 1% (w/v) FDAA in acetone were mixed and heated at 40 °C for 60 min. Then 4 μL of 1 M HCl and 467 μL of 40% (v/v) aqueous acetonitrile were added to the mixture. The latter was then passed through a 0.22-μm filter and subjected to HPLC. The conditions for determining the ee value of L-HPA were as follows: injection volume, 20 μL; mobile phase B (5% (v/v) acetonitrile, 0.05% (v/v) trifluoroacetic acid, and 1% (v/v) methanol); mobile phase C (60% (v/v) acetonitrile, 0.05% (v/v) trifluoroacetic acid, and 1% (v/v) methanol); linear gradient from 0–100%; mobile phase C, 45 min at 30 °C; flow rate, 1.0 mL·min^-1; and UV detection at 340 nm. The L-HPA retention time was 33.65 min.

Homology modeling and molecular docking

Structural homology models of BbPheDH and its mutants were constructed using the SWISS-MODEL server (https://swissmodel.expasy.org/) [49] based on the crystal structure of the PheDH from Rhodococcus sp. M4 (PDB: 1C1D) [35]. The 3D structure of the ligand was obtained from the PubChem website (https://pubchem.ncbi.nlm.nih.gov/) [50]. Ligand energy was minimized with the OPTIMIZE plugin of PyMOL [51]. AutoDock v. 4.2 [52] was used for docking simulations. Prior to docking, each protein was protonated at pH 9.5 using H++ server (http://biophysics.cs.vt.edu/H++) [53] to mimic the experimental condition. All water molecules were removed, and nonpolar hydrogen atoms were added with MGLTools v. 1.5.4. The components of the catalytic triad (K78, K90, and N276) were defined as flexible residues. A grid box 50 × 50 × 50 Å with 0.375 Å spacing encompassed the active cavities of BbPheDH and its mutants and was set as the search space for suitable substrate-binding poses. Energies of the interactions between the ligands and the receptors were calculated with MGLTools v. 1.5.4. The lowest-energy docking pose was selected for the subsequent molecular dynamics simulations.

Tunnel analysis

The tunnels of BbPheDH and its mutant M3-2 were calculated with the CAVER v. 3.0 plugin of PyMOL [54]. The probe radius was set to 1.2 Å, the default settings were applied, and the center of the catalytic triad (K78, K90, D125, and N276) was the starting point. All water molecules were removed, nonpolar hydrogen atoms were added with MGLTools v. 1.5.4, and the volumes of the substrate-binding pockets of BbPheDH and its mutants were calculated with the ProteinsPlus tool (https://proteins.plus/) [55]. The PDB format files were uploaded for the calculations.

L-HPA: L-homophenylalanine. 2-OPBA: 2-oxo-4-phenylbutyric acid. PheDH: phenylalanine dehydrogenase. PPA: phenylpyruvic acid. GluDH: glucose dehydrogenase. HPLC: High Performance Liquid Chromatography.

Ethics approval and consent to participate

Not applicable.

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All authors approved to publish to article.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its Additional file.

Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fundings

This work was supported by Financial support from the National Key Research and Development Program of China (grant numbers 2021YFC2100100), the National Natural Science Foundation of China (NSFC) (grant numbers 21336009 and 21176103), the 111 Project (grant number 111-2-06), the High-end Foreign Experts Recruitment Program (grant number G20190010083), and the National First-Class Discipline Program of Light Industry Technology and Engineering (grant number LITE2018-09).

Author’ contributions

Tao Wu: Conceptualization, Methodology, Investigation, Data curation, Writing-original draft. Xiaoqing Mu: Supervision, Validation, Writing-review & editing, Funding acquisition. Yuyan Xue: Investigation, Validation. Yan Xu: Investigation, Data curation. Yao Nie: Resources, Writing-review & editing, Funding acquisition, Project administration.

Acknowledgements

Not applicable.

Supplementary data

Supplementary data of this work can be found in online version of the paper.

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Structure-Guided Steric Hindrance Engineering of Bacillus Badies Phenylalanine Dehydrogenase for Efficient L-Homophenylalanine Synthesis

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