University of Groningen
Characterization and computation-supported engineering of an ω-transaminase Meng, Qinglong
DOI:
10.33612/diss.172243517
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Meng, Q. (2021). Characterization and computation-supported engineering of an ω-transaminase. University of Groningen. https://doi.org/10.33612/diss.172243517
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39
CHAPTER Ⅱ
Substrate spectrum of an ω-transaminase from
Pseudomonas jessenii analyzed experimentally and
by docking simulations
Qinglong Meng, Cyntia M. Palacio, Henriëtte J. Rozeboom, Dick B. Janssen
Parts of this work were published as:
Palacio CM, Rozeboom HJ, Lanfranchi E, Meng Q, Otzen M, Janssen DB. Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism. FEBS J (2019) 286: 4086–4102.
40
Abstract
A PLP fold-type I ω-transaminase was recently discovered in Pseudomonas jessenii, where it converts 6-aminohexanoic acid to 6-oxohexanoic acid in the caprolactam biodegradation pathway. The substrate scope of this enzyme (PjTA) was probed to identify its potential application as a biocatalyst. In total 34 different substrates were selected including aliphatic amines, aromatic amines, and proteinogenic as well as non-proteinogenic amino acids. The activities towards the selected amino acids were lower than that with the quasi-natural substrate 6-aminohexanoic acid. In the deamination of aromatic amines, PjTA showed significantly higher activity with (S)-1-phenylethylamine than with (R)-1-(S)-1-phenylethylamine, showing that PjTA is an (S)-selective ω-transaminase. Benzylamine was the best substrate for PjTA among a range of aromatic amines including phenylethylamine, while other aromatic amines gave lower activities than (S)-1-phenylethylamine. Regarding the activities with selected aliphatic amines, an increasing trend was identified from short-chain to long-chain amines (ethylamine to 1-aminoheptane). Using crystal structures, interactions between substrate and the active site of PjTA were modeled by docking simulations, which partially explained activity differences with various substrates. The results provide a deeper understanding of substrate recognition by PjTA and the distinct properties of this enzyme in comparison to related ω-transaminases.
41
Introduction
Transaminases are pyridoxal 5′-phosphate (PLP) dependent enzymes that catalyze the transfer of an amino group from a donor (usually an amine or amino acid) to an acceptor (a ketone, aldehyde or keto acid)1. In the resting state, the cofactor is covalently bound to the enzyme via a Schiff base
linkage with a conserved lysine. This structure is called the internal aldimine. The overall transaminase reaction can be divided in two half-reactions, each consisting of several steps (Scheme 1)2. In the first half-reaction, the amino group is transferred to the PLP cofactor via
formation of an imine (external aldimine) and proton shift, leading to a pyridoxamine 5′-phosphate (PMP)-enzyme complex (E:PMP) via two intermediates, a quinonoid and a ketimine. Following the release of the deaminated product as a ketone, aldehyde or keto acid, in the second half-reaction the enzyme binds the amino acceptor and the amino group of PMP is transferred to this acceptor, again via imine formation and proton shift. The aminated product (an amine or amino acid) is released by cleavage of the Schiff base linkage under regeneration of enzyme-bound PLP.
Scheme 1. The mechanism of the transamination reaction catalyzed by ω-transaminases2. Blue arrows
indicate the first half-reaction, green arrows indicate the second half-reaction. All reaction steps are fully reversible.
On the basis of crystal structures, PLP-dependent enzymes are divided into different fold types, subgroups, and classes, and transaminases often belong to fold type I or fold type IV PLP dependent enzymes3−5. The fold type I transaminases consist of L-aspartate transaminases6,
ω-42
transaminases (ω-TAs)9. Fold type IV enzymes include D-amino acid transaminases and
(R)-selective ω-TAs10,11. Transaminases have also been classified in subgroups or classes, mainly
based on substrate type, leading to a classification that only partially agrees with grouping in fold types1,3. The subgroup class I transaminases are active with α-amino acids and thus include the
PLP fold type I aspartate transaminases and branched L-amino acid transaminases. The original class II subgroup, later also called class III transaminases, includes ω-TAs, that is enzymes acting on amino acids with the amino group not at the α position. Many of these enzymes do not require a carboxylate group at all in their substrates. Accordingly, in the second half reaction ω-TAs can accept various carbonyl compounds such as prochiral ketones to synthesize chiral amines, which are important building blocks in the pharmaceutical industry12−14. Enzymes with a broad substrate
scope yet high enantioselectivity and activity are attractive for this purpose. Examples of such enzymes are the well-studied (S)-selective ω-TAs from Ochrobactrum anthropi (OaTA)15,16,
Paracoccus denitrifican (PdTA)17, Chromobacterium violaceum(CvTA)18 and Vibrio fluvialis
(VfTA)19,20. The crystal structures of OaTA (PDB: 5GHF)21, PdTA (PDB: 4GRX)22, CvTA (PDB:
4AH3)23 and VfTA (PDB: 4E3Q)24 were solved. These ω-TAs are dimeric enzymes and the active
sites are located at the interface of the monomers.
In the current study, the substrate scope of the (S)-selective homodimeric ω-TA from
Pseudomonas jessenii (PjTA) was investigated. PjTA is involved in the caprolactam
biodegradation pathway (Scheme 2). Caprolactam is a bulk chemical mostly used for the industrial production of the polyamide nylon 625. Its biodegradation starts with an ATP-dependent
ring-opening reaction that forms 6-aminohexanoic acid (6-AHA). The structure of the enzyme was solved by protein crystallography and the 6-AHA–PLP external aldimine was identified (Figure 1). We now explore the catalytic activity with a broad range of potential amino donors, including various amines and amino acids. We also investigate if computational substrate docking can be used to support structure-based rationalization of the differences in activity obtained with different substrates and between different enzymes. For the latter, we examined the activities of the
structurally related CvTA and VfTA and compare the results with PjTA.
Scheme 2. The route of caprolactam degradation in Pseudomonas jessenii converges to 6-AHA, which is
43
Figure 1. The crystal structure of the 6-AHA–PLP (cyan–magenta) external aldimine adduct (PDB: 6G4E)
of PjTA, with Arg417 forming a bidentate salt bridge to the carboxylate of 6-AHA. Residues forming the active site are displayed as sticks in tan color, Phe86', Ser87', and Thr324' from the other subunit in light green. Lys287 in dark green. Black dotted lines with distances in Å indicate hydrogen bonds.
Materials and methods
Materials
Nicotinamide adenine dinucleotide (NAD) and alanine dehydrogenase from Bacillus cereus (BcAlaDH) were purchased from Sigma-Aldrich (Zwijndrecht, the Netherlands). Amines and amino acids were purchased from Sigma-Aldrich or Acros Organics. PLP was purchased from Fisher Scientific.
Enzyme expression and purification
PjTA was produced in E. coli strain BL21 (DE3) using the PjTA gene cloned in-frame
downstream of the hexahistidine tag sequence in expression vector pET-20b(+). After inoculation and overnight growth of precultures, the cells were transferred to 500 mL TB medium with 100 µg/mL ampicillin and cultures were shaken at 37 °C. When the OD600 value reached 0.6, the
temperature was lowered to 24 °C and induction was started by addition of 1 mM of IPTG. After 16 h of growth the cells were collected at 7,000 g for 30 min and the pellets were washed and suspended in 20 mM Tris-HCl, 500 mM NaCl, 20 µM PLP, pH 7.5. After sonication (15 min, 5 s intervals), the lysate was centrifuged at 36,000 g for 45 min at 4 °C. The supernatant was loaded onto a 5 mL HisTrap column (GE Healthcare, Sweden) and the enzyme was isolated with an AKTA purifier using a linear gradient of 0 to 500 mM imidazole in 20 mM Tris-HCl, 500 mM NaCl, 20 µM PLP, pH 7.5. Fractions containing the enzyme as judged by SDS-PAGE were pooled and desalted in phosphate buffer (50 mM potassium phosphate, 20 µM PLP, pH 8.0) with Econo-Pac 10DG columns (Bio-Rad, USA). The purity was checked by SDS-PAGE, and the protein was
44
quantified with the Bradford assay. Purified enzymes were stored in aliquots at −80 °C in phosphate buffer.
Activity assays
The activity of PjTA coupled to pyruvate amination was measured using a coupled assay by following the alanine-dependent reduction of NAD+ by alanine dehydrogenase at 340 nm (ε
NADH =
6.22 × 103 M−1·cm−1)26. Excess BcAlaDH was included in the reaction mixtures to ensure that the
rate-limiting step is the alanine-forming transamination reaction. The pyruvate concentration was optimized to ensure high BcAlaDH activities and a minimal lag phase. The reaction mixtures contained 15 mM substrate, 2 mM NAD+, 0.05 mM PLP, 5 U/mL BcAlaDH, 0.015 mg/mL PjTA,
and 0.2 mM pyruvate in 100 mM potassium phosphate, pH 8.0. Assays were performed in flat-bottomed 96-well microtiter plates. The absorbance at 340 nm was monitored during 30 min at 30 °C using a microtiter plate reader (Synergy Mx Microplate Reader; BioTek Instruments, Winooski, VT, USA). The plates were prewarmed and reactions were started by addition of 150 µL of 0.4 mM pyruvate solution to 150 µL reaction mixture. All assays were done in triplicate. The initial rates of the reduction of NAD+ observed with different substrate concentrations were used to
determine the kinetic constants. Specific activities were expressed in units per mg of protein (µmol·min−1·mg−1).
Substrate docking
Docking of substrate structures in the PjTA active site was done with Yasara (version 20.4.24). To prepare the crystal structure of PjTA (6G4D) for docking, the waters were removed and the hydrogen bonding network was optimized using Yasara. The cofactor PLP was kept in the structure. After adding missing hydrogens, an energy minimization was applied to the PjTA structure using the AMBER03 force field. Structures of the substrates were drawn with ChemDraw 3D (version 16.0.1.4), and, missing hydrogens were added with Yasara. Covalent docking of the external aldimine was performed using the Yasara "dock_runcoval.mcr" routine, with formation of a covalent bond between the PLP cofactor and the amine substrate. The docking poses with the lowest energy were selected for visual inspection and realistic positions and interactions of the substrate were analyzed.
Results and discussion
Substrate spectrum of PjTA
In view of the importance of a wide substrate scope for use of ω-TAs in applied biocatalysis, the selectivity of PjTA was examined with a range of substrates. For convenience, we used a coupled enzyme assay in which L-alanine is used as the amine donor and pyruvate formation is detected by coupling to NADH oxidation via alanine dehydrogenase. In total 33 amines and amino acids including both aliphatic and aromatic compounds were examined (Table 1).
45
Although 6-AHA is not known to occur naturally27, it can be considered as the biologically
most relevant substrate for PjTA since the Pseudomonas strain from which the enzyme originates was enriched by selection for growth with caprolactam as the sole carbon source. The results with other substrates are therefore expressed in comparison to 6-AHA. Unsurprisingly, the results showed that 6-AHA was one of the best substrates for PjTA, together with the structurally similar amino acids 5-aminopentanoic acid (5-APA) (95% of the 6-AHA activity) and 4-aminobutanoic acid (4-ABA) (129%).
Of the aromatic amines, (S)-1-phenylethylamine [(S)-PEA] gave the same activity as 6-AHA whereas (R)-1-phenylethylamine [(R)-PEA] exhibited 17% activity, suggesting modest enantioselectivity. However, in the reverse direction, the latter reaction allowed reduction of acetophenone with the enantiomer excess of (S)-PEA reaching >99%28. Since activities reported in
Table 1 are obtained at a single fixed substrate concentration, effects of differences in KM are not
apparent. PjTA slightly converted (R)-PEA probably because the phenyl group of (R)-PEA can be marginally accepted by the small binding pocket of PjTA. In view of the observed
enantioselectivity, PjTA can be considered as an (S)-selective ω-TA, in agreement with the phylogenetic classification of PjTA as a class Ⅲ, PLP-fold-type I transaminase3. Nevertheless, the
(S)-enantioselectivity of PjTA in the kinetic resolution of PEA is lower than that of some related fold-type I transaminases such as an ω-TA from Halomonas elongata (HeTA) for which the relative activities with (S)- and (R)-PEA were higher than 20:129. Also CvTA18, VfTA19, and an
ω-TA from Burkholderia vietnamiensis G4 (Bvω-TA)30 converted (S)-PEA while exhibiting no
detectable activity with (R)-PEA.
PjTA displayed 2-fold higher activity with benzylamine than with (S)-PEA. The absence of
the methyl group in benzylamine apparently causes better substrate accommodation, suggesting that the pocket that accommodates the additional methyl group of (S)-PEA is not optimally accommodated in the small pocket. With most other aromatic amines, PjTA exhibited lower activities than with (S)-PEA. Also with all aliphatic amines tested, activities were lower than with (S)-PEA, and a downward trend of activity was visible going from long-chain (1-aminoheptane) to short-chain amines (ethylamine). For the aliphatic diamines, no activity was detected with PjTA, indicating that a negatively charged substituent in the rest group is much better accepted than a positively charged group.
In comparison to activity with 6-AHA, PjTA also showed poor activities with the α-amino acids that were tested, and most of them gave no detectable activity at all. PjTA showed some activity with L-ornithine (14% of the 6-AHA activity) and L-2,4-diaminobutyric acid, but not with D-ornithine and D-2,4-diaminobutyric acid, confirming the (S)-selectivity of the enzyme.
The activity of some other well-studied (S)-selective homodimeric ω-TAs has been explored earlier. This includes the CvTA and VfTA18-20, which share 40% and 42% sequence
identity with PjTA, respectively. The substrate scopes of CvTA and VfTA have been expanded by protein engineering to improve activity towards bulky amines31,32 , aliphatic amines33,
46
Table 1. Activities of PjTA, CvTA, and VfTA with various amino donors.a
Specific activity (%)
Substrate Structure PjTA CvTA VfTA
6-AHA 100 (0.9)b 180 35 (S)-PEA 100 100 (1.6)c 100 (1.0)c (R)-PEA 17 _d _ 5-APA 95 93 4 4-ABA 129 174 70 benzylamine 208 406 208 2-phenylethylamine 28 201 106 3-phenylpropylamine 25 77 60 4-phenylbutylamine 99 145 28 3-fluorobenzylamine 76 82 54 4-fluorobenzylamine 67 95 79 1-aminoheptane 68 _ _ 1-aminohexane 52 _ _ 1-aminopentane 43 _ _ 1-aminobutane 21 _ _ 1-propylamine 10 _ _ ethylamine <1 _ _ spermidine <1 _ _ 1,4-diaminobutane <1 _ _ 1,3-diaminopropane <1 _ _ glycine 6 _ _ L-serine 3 11 4 L-ornithine 14 _ _ D-ornithine <1 _ _ L-2,4-diaminobutyric acid 7 _ _ D-2,4-diaminobutyric acid <1 _ _
47
a Pyruvate was used as the amino acceptor and substrates were add at 15 mM. Assay details are given in Materials and
Methods. All activity data are averages from triplicate assays. No activity (<1%) was also found with the amino acids L-Leu, L-Phe, D-Phe, L-Lys, D-Lys, L-Glu, D-Glu, and β-alanine. b The specific activity of PjTA with 6-AHA was 0.9
U/mg (1 U equals 1 µmol/min) and set as 100% relative activity. c The specific activities of CvTA and VfTA were 1.6
and 1 U/mg, respectively and set as 100% relative activity in their respective substrate profile. d Not determined.
To further compare the activities of PjTA with those of CvTA and VfTA, coupled transaminase activity assays were done with pyruvate as the acceptor substrate and results are reported with (S)-PEA set as the reference substrate (Table 1). In the deamination of (S)-PEA, the specific activities of CvTA and VfTA were higher than that of PjTA. For the activity towards other aromatic amines,
CvTA always displayed the best performance among those three transaminases. Furthermore, CvTA also gave a higher activity comparing the other two transaminases when converting 6-AHA
and 4-ABA.
As with PjTA, the activity towards benzylamine of CvTA and VfTA was higher than that with (S)-PEA, in agreement with Kaulmann et al.18 and Shin and Kim19 who also observed that
activities of CvTA and VfTA with benzylamines were higher than with PEA, while other (S)-selective dimeric ω-TAs such as BvTA and HeTA showed lower activities towards benzylamine than with (S)-PEA29,30. The activities of CvTA towards 6-AHA and 4-ABA reported by Kaulmann
et al.18 are lower than that towards (S)-PEA, which is opposite to that we observed. Low or even no
detectable activities towards α-amino acids such as L-serine and L-leucine were found with ω-TAs including PjTA, CvTA , VfTA, BvTA, and HeTA18,19,29,30. The activity of PjTA with β-alanine was
almost not detectable, which was also the case with in CvTA, VfTA, HeTA, and BvTA18−20,29,30.
This clearly distinguishes these transaminases from the homologous PLP fold-type I β-amino acid transaminases discovered in bacterial cultures enriched with β-amino acids as growth substrate8.
Docking analysis
Different structures of PjTA were recently solved by protein crystallography, including an
E∙succinate complex (PDB: 6G4B), an E∙phosphate complex (6G4C), an E∙PLP complex (6G4D), an E∙PLP–6-AHA intermediate (6G4E) (Figure 1) and an E∙PMP complex (6G4F). In view of the differences in activity of PjTA with various substrates and the availability of the crystal structure of the external aldimine, we examined if the activity profile of PjTA can be understood by molecular docking, and whether there is a structural explanation for differences in activity profiles of PjTA,
VfTA and CvTA.
First, as a control, the structure that was obtained by covalent docking of 6-AHA as the ligand and PjTA with bound PLP as the receptor was compared to the crystal structure of the external aldimine (PDB: 6G4E). This allowed a comparison between the structure predicted by covalent docking and the experimental crystal structure, revealing the accuracy of the docking simulation. The docked structure indeed was about the same as the crystal structure (Figure 2), suggesting the docking protocol delivered an accurate result. Structures of the 6-AHA external aldimine intermediates were also constructed for CvTA and VfTA, respectively, which revealed
48
that these proteins have phenylalanine side chains (Phe89 and Phe85, respectively) pointing towards a conserved arginine (Arg416 in CvTA, Arg415 in VfTA) that restricts space at the active site entrance (Figure 3A). In PjTA the amino acid at the corresponding position is the smaller Ser87. The resulting more spacious active site entrance allows for repositioning of Arg417 to a site where it can form a salt bridge with the carboxylate group of 6-AHA. The dual interaction of Arg417 with both the carboxylate of the substrate and Ser87 indicates better active site geometry for 6-AHA conversion in PjTA. In line with this finding, previous studies using VfTA revealed that its substrate scope can be altered or expanded by protein engineering of the residue
Arg41524,31,33,35−37. The result of the docking analysis is consistent with the relative transaminase
activities with 6-AHA observed by other researchers, while in our study the activity of CvTA towards 6-AHA was higher than that of PjTA.
A comparison of the docked structures of these three enzymes (PjTA, VfTA, and CvTA) with 4-ABA suggested the same feature (free vs. restricted movement of the conserved switching arginine) causes the high activity of PjTA towards 4-ABA (Figure 3B), which is consistent with results found by others. Nevertheless, the activity of PjTA with 4-ABA is still lower than that of
CvTA according to our observation.
The slightly higher activity of PjTA in the deamination of 4-ABA in comparison to the activity with 6-AHA can be attributed to different interactions in the entrance tunnel. Since the shorter carboxylic acid moiety of 4-ABA results in a longer distance to Arg417, Arg417 may move further inward to the carboxylate group of 4-ABA to preserve the salt bridge. Then, the distance between the Arg417 iminium group from one monomer and the Ser87 OH from the other monomer becomes longer, causing loss of the hydrogen bond between these groups, facilitating interaction between the iminium group and the 4-ABA carboxylate bound in the tunnel (Figure 4).
PjTA showed almost the same activity towards 5-APA as that with 6-AHA probably because the
distance between Arg417 and the carboxylate group of 5-APA is adequate to keep their salt bridge without the movement of Arg417 (Figure 4).
Figure 2. Comparison of the crystal structure (6G4E) (cyan) and the docked structure (6G4D) (orange) of
49
Figure 3. Docked structures of the external aldimine forms of 6-AHA (A) and 4-ABA (B) in PjTA, CvTA,
and VfTA. PjTA in cyan, VfTA in orange, and CvTA in magenta.
Figure 4. The docked structure of the external aldimine complexes of three ω-amino acids in PjTA. 6-AHA
in cyan, 5-APA in orange, and 4-ABA in magenta.
Primary arylalkylamines with different aminoalkyl groups on the aromatic ring (benzylamine, 2-phenylethylamine, 3-phenylpropylamine, 4-phenylbutylamine) gave varying activities with PjTA when tested as amino donors. Among these four amines, benzylamine exhibited the best activity, while 2-phenylethylamine and 3-phenylpropylamine were converted much slower, probably because residues Arg417, Leu57 and Trp58 gave steric hindrance to the phenyl group of the latter two amines (Figure 5A). Intriguingly, the activity towards
50
suggesting some favorable interactions between the Phe moiety of 4-phenylbutylamine and residues in the large binding pocket or tunnel leading to it (e.g. Tyr20, Gly56, Leu57, Trp58, Phe86, and Ala230), while residue Gly56 is not involved in a hydrophobic interaction with 3-phenylpropylamine or 2-phenylethylamine.
Furthermore, PjTA displayed lower activities towards benzylamines with a fluorine substituent at different positions of the phenyl group (3-fluorobenzylamine and
4-fluoro-benzylamine) than that with benzylamine. Benzylamine could have a hydrophobic interaction with Tyr20, Leu57, Trp58, Phe86, and Ala230, which we supposed to be important for activity.
Suspectedly, only Leu57, Trp58, and Ala230 have a similar hydrophobic interaction in case of 3-fluorobenzylamine or 4-3-fluorobenzylamine because the fluorine group may form a hydrogen bond with Arg417 and Trp58, causing the phenyl ring to be locked in a different position, with loss of the hydrophobic interaction on the opposite side of the ring with Tyr20 and Phe86 (Figure 5B).
Figure 5. The active site of PjTA with docked external aldimines. Benzylamine in cyan. A,
2-phenylethylamine in orange, phenylpropylamine in magenta, 4-phenylbutylamine in gray. B, 3-fluorobenzylamine in orange, 4-3-fluorobenzylamine in magenta. Black dotted lines with distances in Å indicate hydrogen bonds.
Being less bulky than aromatic amines, the activities of PjTA towards aliphatic amines will mainly depend on the hydrophobic interaction between the alkyl moiety and hydrophobic residues (e.g. Gly56, Leu57, Trp58, Phe86, and Ala230) (Figure 6). For longer aliphatic amines the
hydrophobic interaction could be stronger. For example, 1-aminoheptane with the longest chain gave the highest activity and residues Gly56, Leu57, Trp58, Phe86, and Ala230 can form a hydrophobic cup that likely interacts with the heptyl moiety of 1-aminoheptane, while ethylamine with the shortest chain may have only a hydrophobic interaction with Ala230 (Figure 6). PjTA exhibited no detectable activity towards aliphatic diamines such as 1,4-diaminobutane and
1,3-51
diaminopropane, whereas the activities with their corresponding aliphatic amines 1-aminobutane and propylamine were higher. According to Figure 7, when 1,4-diaminobutane or
1,3-diaminopropane binds to the active site, the amino group of the substrate may push Arg417 away and allow Arg417 and Ser87 to form a stronger hydrogen bond between the two monomers, and thereby it could partially hinder substrate access or product exit through the tunnel.
Figure 6. The active site of PjTA with docked external aldimines formed from n-alkylamines. Residues
shaping the hydrophobic cup are displayed as sticks. Aminoheptane in cyan. Aminohexane in orange. 1-Aminopentane in light green. 1-Aminobutane in magenta. 1-Propylamine in light blue. Ethylamine in tan.
Figure 7. The active site of PjTA with docked external aldimines. (A) 1-Aminobutane in cyan,
52
Conclusions
The activity data presented here show that PjTA has a wide substrate range in the deamination of aliphatic and aromatic amines. Activities with L-amino acids are absent or low, whereas ω-amino acids are well accepted. A distinct feature of PjTA is the presence of a Phe to Ser close to the entrance of the tunnel leading to the active site. This could play a role in the adaptation of the enzyme to 6-aminohexanoic acid deamination, as the Ser allows more space for an outward movement of the switching arginine, yet maintaining polar interactions. The same effect could contribute to the good activity with other substrates in which a group that interacts with the
arginine iminium is far away from the reactive amine group. Computational substrate docking was a useful tool to obtain an understanding of such structural features that contribute to activity differences of PjTA with various substrates. Small differences in hydrophobic interactions within the tunnel leading to the active site can also cause differences in activity between a series of aliphatic and aromatic substrates.
Author contributions
QM performed the activity assay and computational docking analysis and wrote the corresponding part of the manuscript. CMP cloned the enzyme, performed kinetic experiments, and wrote the manuscript. HJR solved the crystal structures and wrote the manuscript. DBJ revised the manuscript and supervised the project.
53 References
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