D-Phenylglycine aminotransferase (d-PhgAT)-substrate scope and structural insights of a stereo-inverting biocatalyst used in the preparation of aromatic amino acids

Article abstract of DOI:10.1039/d0cy01391a

Enantiopure amines are key building blocks in the synthesis of many pharmaceuticals, so a route to their production is a current goal for biocatalysis. The stereo-inverting d-phenylglycine aminotransferase (d-PhgAT), isolated from Pseudomonas stutzeri ST-201, catalyses the reversible transamination from l-glutamic acid to benzoylformate, yielding α-ketoglutarate and d-phenylglycine (d-Phg). Detailed kinetic analysis revealed a range of amine donor and acceptor substrates that allowed the synthesis of enantiopure aromatic d-amino acids at a preparative scale. We also determined the first X-ray crystal structure of d-PhgAT with its bound pyridoxal 5′-phosphate (PLP) cofactor at 2.25 ? resolution. A combination of structural analysis and site-directed mutagenesis of this class III aminotransferase revealed key residues that are potentially involved in the dual substrate recognition, as well as controlling the stereo-inverting behaviour of d-PhgAT. Two arginine residues (Arg34 and Arg407) are involved in substrate recognition within P and O binding pockets respectively. These studies lay the foundation for further enzyme engineering and promote d-PhgAT as a useful biocatalyst for the sustainable production of high value, aromatic d-amino acids. This journal is

Full text of DOI:10.1039/d0cy01391a

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DOI: 10.1039/D0CY01391A
ARTICLE
Received 00th January D-phenylglycine aminotransferase (D-PhgAT) – substrate scope and structural insights of a stereo-
0xx, inverting biocatalyst used in the preparation of aromatic amino acids.
Accepted 00th January Annabel Serpico[a/d], Silvia De Cesare , Jon Marles-Wright , M. Kalim Akhtar , Gary Loake , Dominic J.
0xx
Campopiano*[a]
2
[a]
[b]
[c/e]
[c]
2
DOI: 10.1039/x0xx00000x Enantiopure amines are key building blocks in the synthesis of many pharmaceuticals, so a route to their
production is a current goal for biocatalysis. The stereo-inverting D-phenylglycine aminotransferase (D-
PhgAT), isolated from Pseudomonas stutzeri ST-201, catalyses the reversible transamination from L-
glutamic acid to benzoylformate, yielding -ketoglutarate and D-phenylglycine (D-Phg). Detailed kinetic
analysis revealed a range of amine donor and acceptor substrates that allowed the synthesis of enantiopure
aromatic D-amino acids at a preparative scale. We also determined the first X-ray crystal structure of D-
PhgAT with its bound pyridoxal 5’-phosphate (PLP) cofactor at 2.25 Å resolution. A combination of structural
analysis and site-directed mutagenesis of this class III aminotransferase revealed key residues that are
potentially involved in the dual substrate recognition, as well as controlling the stereo-inverting behaviour of
D-PhgAT. Two arginine residues (Arg34 and Arg407) are involved in substrate recognition within P and O
binding pockets respectively. These studies lay the foundation for further enzyme engineering and promote
D-PhgAT as a useful biocatalyst for the sustainable production of high value, aromatic D-amino acids.
Introduction
The non-proteinogenic amino acids D-phenylglycine (D-Phg) and
D-4-hydroxyphenylglycine (D-Hpg) are fundamental side chain
building blocks in many antimicrobial compounds, including the
amoxicillin and vancomycin group of antibiotics 1,2. Existing
synthetic methods for the preparation of D-Phg and its derivatives
rely on toxic cyanide chemistry and multi-step processes 3.
Alternative biocatalytic methods are limited by substrate solubility
(
e.g. the coupled hydantoinase/carbamoylase system), or rely on a
D-amino acid aminotransferase that uses the costly D-amino acid
donor and requires an additional enzyme (a racemase) to yield the
4
,5
desired product
. Aminotransferases (ATs, also known as
6
transaminases, TAs or ATAs, EC 2.6.1) are a large superfamily of
pyridoxal 5’-phosphate (PLP)-dependent enzymes with a rapidly
expanding range of industrial applications for the production of
diverse compounds containing a chiral amine functionality 7–15. For
example, they have been shown to be suitable in the replacement
of transition metal catalysts for the preparation of drugs such as
sitagliptin 16. ATs use PLP to catalyze the transfer of an amino
group between an amino donor and an amino acceptor via a ping-
pong, bi-bi mechanism via a pyridoxamine (PMP) form of the
cofactor (Scheme 1).
Scheme 1. Simplified, two-step mechanism of the D-
PhgAT-catalysed reaction. In step 1, L-Glu donates the
amino group to the pyridoxal 5’-phosphate (PLP) cofactor
generating pyridoxal amine (PMP) and α-ketoglutarate (AKG).
In step 2, benzoyl formate (BZF, R = H) or its 4’-hydroxy
derivative (HBF, R = OH) accepts the amino group from PMP
yielding D-Phg or D-Hpg respectively 11. L-Glu structure fixed.
The 3D structures of many PLP enzymes have been determined
and they display seven different folds with TAs typically found in
classes I and IV 17–22. To determine the substrate scope of so many
AT-catalysed reactions, powerful high-throughput assays have
The X-ray structures and screening methods for these biocatalysts
have been comprehensively reviewed 6 but there are many ATs
that display “unusual” characteristics which require further study.
been developed that can be used to study the activities of wild-type
ATs or identify new substrates for engineered enzymes 23–27.
[
a] School of Chemistry, University of Edinburgh, Joseph Black Building, David
Brewster Road, Edinburgh, EH9 3FJ (UK) E-mail:
Dominic.Campopiano@ed.ac.uk. [b] School of Natural and Environmental
Sciences, Newcastle University, Newcastle upon Tyne, NE1 7RX (UK).
[c] Institute for Molecular Plant Sciences, School of Biological Sciences,
University of Edinburgh, King’s Buildings, Edinburgh EH9 3BF (UK). Current
address: [d] LEITAT Technological Center, Terrassa (Barcelona), Spain and [e]
Department of Chemistry, United Arab Emirates University, Al Ain (SAU).
†
Electronic
Supplementary
Information
(ESI)
available:
See
DOI: 10.1039/x0xx00000x
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The D-phenylglycine aminotransferase (D-PhgAT), isolated from using
a modified Chirobiotic-T chiral HPLC method and
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the soil bacterium Pseudomonas stutzeri ST-201 28,29catalyses the demonstrated the enzyme enantioselectivit Dy Ow I :i t 1h0 . p1 0r o3 9d /uDc 0t iCo Yn 0 1o 3f 9 D1 A-
reversible transamination of L-glutamic acid (L-Glu) with the amino Phg in >98% e.e. (Figures S6 and S7) 36. Furthermore, we used
acceptors benzoylformate (BZF) and 4-hydroxy benzoylformate this method to determine the D-PhgAT kinetics, which match well
(
HBF) yielding the R-enantiomers D-Phg and D-Hpg respectively. with the coupled assay (Figure S8 and Table S1). With BZF as the
The keto product derived from L-Glu is -ketoglutarate (AKG) amino acceptor the chiral HPLC assay was used to screen D-Phg
Scheme 1). What is unique to this AT is its so-called “stereo- production with various commonly used amino donors. These
(
inverting” characteristic, where the amino donors (L-Glu and D- included L- and D-amino acids, R- and S-methylbenzylamine
Phg) in this reversible reaction exhibit inverse absolute (MBA), isopropylamine (iPrA), as well as o-xylene diamine (OXD))
configurations. Since it uses an inexpensive L- (or S-) amino acid (Figures S9 and S10). The production of D-Phg is observed with
donor the P. stutzeri D-PhgAT represents an attractive biocatalyst several amino donors (apart from iPrA) and the activity is ranked
for the synthesis of enantiomerically pure D-Phg (or R-Phg) relative to L-Glu as the best amino donor. This broad utility is in
derivatives in a single step 5. D-PhgAT has also been used in contrast with a previous report which observed a very limited
qualitative and quantitative analysis of L-Glu concentrations in food substrate scope 28. Regardless of the chirality of the amino donor,
3
0
when coupled with L-glutamate dehydrogenase
and for quality the enantiopurity of the final D-Phg product is not affected. Since L-
control of amoxicillin in pharmaceuticals when coupled with a Glu was the best amino donor it was used to scale up the reaction
penicillin acylase 31. The homologous D-PhgAT from P. putida 32–34 with 1 g BZF substrate. Using D-PhgAT (1 mg mL ) 93%
-1
(
82% amino acid sequence identity) has been successfully conversion to D-Phg (99% e.e.) was observed after ~3 h (Figure
combined with hydroxymandelate synthase (HmaS) and S11). We purified the D-Phg product by preparative HPLC (Figure
hydroxymandelate oxidase (Hmo) to engineer an E. coli strain that S12) and characterized it using 1H and 13C NMR and ESI-MS
is able to produce D-Phg 34.
(Figure S13) which are identical to an authentic standard.
To expand its potential as a valuable biocatalyst, comprehensive We also tested the promiscuity of the P. stutzeri D-PhgAT towards
substrate and structural analyses of the P. stutzeri D-PhgAT are aromatic acceptors. The goal here was that these would yield
required. An understanding of the mechanism of its unique stereo- various D-aromatic amino acids since D-Phe, D-Trp and D-Tyr are
inverting activity will also aid in increasing its substrate scope by used as chiral building blocks for many clinically useful drugs 37–39.
enzyme engineering 35. In this study we describe the isolation of
recombinant P. stutzeri D-PhgAT and, by a combination of
spectrophotometric and chiral product analysis, we explore the
amino substrate range and enantioselectivity of this enzyme. This
revealed a broader substrate range than previously anticipated,
which led us to use this biocatalyst to prepare a range of aromatic
D-amino acids in high conversion and e.e. Furthermore, we
determined the first X-ray structure of the PLP-bound form of the P.
stutzeri D-PhgAT at 2.25
Å resolution, which confirms its
classification as a member of the class III TA family. To explore the
dual substrate binding mechanism, we then compared the D-
PhgAT structure with the PLP-bound, external aldimine complexes
of similar TAs. Furthermore, a combined sequence and structural
analysis led to site-directed mutagenesis studies which identified
potential residues required for catalytic activity and substrate
recognition. This work lays the foundation for future enzyme
engineering, and strengthens the utility of D-PhgAT as an important
biocatalyst for the preparation of enantiopure D-aromatic amino
acids. Shortened this last paragraph.
Results and discussion
Recombinant, codon optimized, P. stutzeri D-PhgAT was produced
in E. coli BL21 (DE3) with an N-terminal 6xHis affinity tag. It was
purified to homogeneity in high yield by standard nickel affinity and
gel-filtration chromatography methods, which showed it to be
homodimeric in solution (Figure S1). The enzyme shows a distinct
UV/visible spectroscopic profile with a max at 411 nm, indicative of
PLP binding; this changes upon addition of the amino donor L-Glu
and leads to PMP formation which absorbs at 311 nm (Figure S2).
A convenient, high-throughput, coupled assay used the -
ketoglutarate dehydrogenase (AKGDH) enzyme to monitor
transamination of L-Glu through NADH production (Figure S3, S4).
This assay allowed the determination of kinetic parameters
Scheme 2. D-PhgAT reactions for the synthesis of
aromatic D-amino acids from L-Glu and aromatic amino
acceptors.
We used D-PhgAT with L-Glu and the five amino acceptors (HBF,
BZF, indole pyruvic acid (IPA), phenylpyruvic acid (PPA), and 4-
hydroxyphenylpyruvic acid (HPPA)) that would yield the
corresponding aromatic amino acids D-Hpg, D-Phg, D-Trp, D-Phe,
and D-Tyr (Scheme 2). The enzyme shows good affinity towards all
(
apparent K
mM respectively) in the “forward” D-amino acid producing direction
Figure S5, Table S1). We confirmed the chirality of the product
M
for L-Glu and BZF are 9.85 ± 0.62 mM, 1.81 ± 0.57
(
8
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acceptor substrates tested (apparent K
IPA to 9.24 mM for HPPA, Table 1).
M
values from 0.79 mM for of group III of the aspartate aminotransferase family, which falls
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4
1
within the type I PLP-dependent superfamil yD Of oI :l d1 0.1 .0 39/D0CY01391A
The final refined model contains three homo-dimers of D-PhgAT
with the PLP cofactor present as an internal aldimine bound to
residue Lys269 in each chain (Figures 2, S14 and S15). Each
monomer can be subdivided into two distinct domains: a small
discontinuous domain comprising the residues 1-72 and 336-453;
and a large domain formed by residues 73-335 (Figure S14). The
N-terminal part of the small domain comprizes a kinked -helix
followed by a three-stranded antiparallel -sheet, while the C-
terminal part consists of an -helix followed by two antiparallel
Substrate
HBF
Conversion %
50
ee %
K
M
(mM)[c]
kcat/K
M
(M-1 s-1)
>98
1.01 ± 0.07
3.16 ± 0.46
0.39 ±0.19
6.91± 0.99
9.24 ± 2.40
899.15 ± 0.27
302.80 ± 0.10
262.12 ± 0.13
14.49 ± 0.82
0.06 ± 0.01
BZF
IPA
93
30
57
15
>98
>98
>98
>98
PPA
HPPA

strands, an extended loop interspersed with a short -helix and
continues into a longer-helix. This is followed by a -strand that
extends the N-terminal -sheet and an additional -strand, which
extends the C-terminal -sheet, the structure finishes with an -
helix opposed between the two other major helices in this domain.
The large domain consists of a central seven-stranded -sheet,
with five parallel strands and two in an anti-parallel orientation. The
Table 1. Analysis of the D-PhgAT kinetic parameters with
various amino acceptors. Kinetic parameters of D-PhgAT for
five amino acceptors, obtained using the chiral HPLC method.
Reactions were carried out for 15 minutes using 100 mM L-Glu
and different amino acceptor concentrations (0-40 mM). To
determine % conversions, reactions were carried out at 100
mg scale as described in Material and Methods.

-sheet is connected by -helices, which harbor the PLP cofactor
binding site and main dimerization interface (Figure S14).
The essential Lys269 is located in the loop that connects strands
Furthermore, we found again that the conversion was highly
stereoselective with only the D-enantiomer produced (Figure 1).
Kinetic parameters and % conversions were calculated using the
chiral HPLC method and are summarized in Table 1. Using this
data larger scale biotransformations were carried out at up to 100
mg scale for each amino acceptor. The D-PhgAT displayed modest
to excellent conversion for these aromatic acids, with the highest
observed for the BZF to D-Phg conversion (93%). This analysis

9 and 10, with the aromatic ring of the PLP sandwiched between
Val243 and Tyr149 (Figure S15). Water molecules (red spheres)
form bridges between the PLP phosphate oxygens and the side
chains of Glu124, Thr303 and the backbone of Phe304 from the
opposite monomer. The phosphate is also coordinated by H-bonds
to Ser121, Gly122 and Thr123 from the same monomer 42.
Since the ATs bind two amino-acid substrates, it has been
proposed that this dual-substrate recognition is accommodated in
two binding sites. These were named the O-pocket (or O-site,
defined by the proximity of residues to the 3’-O of the PLP) and P-
pockets (or P-site, defined by the proximity to the PLP-phosphate)
by Dijkstra and colleagues in their study of the S-selective TA
(
MesAT) 43. They determined the structures of this useful enzyme
with the PLP-external aldimine forms of S--Phe and R-3-
methylhexanoic acid, as well as with the amino acceptor AKG.
These complexes allowed them to identify residues in the binding
pockets as well as a pair of key arginine residues (Arg54 and
Arg412) in this type I enzyme. The O- and P-pocket hypothesis was
also explored by a comprehensive structural and sequence
analysis of the AT superfamily recently carried out by Bornscheuer
and colleagues to identify 13 key residues (including specific
arginines) that they propose as crucial in controlling substrate
binding and reaction specificity 11. Bornscheuer and others have
also put forward an elegant model known as the ‘arginine flip or
switch’ that is thought to control the dual specificity of these
enzymes 4 . By modelling into the incomplete apo D-PhgAT
structure (2CY8) they suggested that a key Arg residue, equivalent
to Arg407 in our structure, binds to the -carboxylate of the L-Glu
donor and may switch in and out of the active site. A recent paper
by Walton et al also determined the crystal structure of D-PhgAT
(PDB code: 6DBS) and they used this to propose a two pocket
Figure 1. Chiral HPLC traces of the synthesis of
enantiopure D-Tyr, D-Phe, D-Trp, D-Phg and D-Hpg.
Reactions described in Scheme 2 were analysed for product
formation by chiral HPLC using a Chirobiotic T column with
monitoring at= 205 nm.
4–47
emphasizes the broad utility of the enzyme.
To understand the molecular basis of the substrate promiscuity and substrate binding model and residues involved in catalysis 48.
the origin of the D-enantioselectivity, as well as revealing the Unfortunately, like 2CY8, the D-PhgAT had no PLP bound (but
residues involved in catalysis, we determined the crystal structure instead had phosphate) and lacked defined electron density for
of the D-PhgAT in complex with the PLP-cofactor bound as the important residues 28-36 and 292-302 42,48. These incomplete apo-
internal aldimine form at 2.25 Å resolution (Figure 2, S14 and S15). and phosphate-bound structures are missing important features
Here we used the incomplete structure of the P. stutzeri enzyme
lacking the bound PLP cofactor (PDB code: 2CY8 40) as a
molecular replacement model (Table S2, PDB code: 6G1F). The
overall D-PhgAT structure confirmed its classification as a member
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such as the P-pocket. In contrast, with the PLP VbieowuAnrdticleinOnoliuner
structure we can now better define both p oD c Ok Ie: t1 s0 . f1 o0 r3 9t h/ De 0 fCi rYs 0t 1 t3 i 9m 1 eA
(
Figure 2B). The O-pocket is formed by the side chains of Phe63,
His66, His213, Phe304, and Arg407. On the other side of the PLP
cofactor the P-pocket is formed by residues Arg34, Gln301, and
Thr303.
The enzyme is unusual in that it can bind both L- and D- forms of
the amino acid substrates (e.g. L-Glu and D-Phg) depending on
which reaction (forward or reverse) it is catalyzing (Schemes 1 and
2). This inherent substrate promiscuity of the enzyme and the
product enantioselectivity appears to be due to a combination of
these two pockets (Figure 2B) and a cavity (Figure 2C) that can
accommodate large hydrophobic amino acceptors. This cavity
extends from the surface of the protein and has a wide mouth with
a constriction near the active site formed by the side chains of
His66 and His213. This constriction accommodates hydrophobic
substrates through pi-stacking interactions, while orienting the keto
substrate to accept the amine group from the PMP intermediate.
This results in the formation of the key PLP:substrate external
+
aldimine intermediate that undergoes the enantiomeric H transfer
at C4’ to form the D-product (Figure S16). We were also struck by
the pair of arginine residues provided by one of the subunits, Arg34
(
in the P-pocket) and Arg407 (in the O-pocket) (Figure 2B). In the
PLP bound structure, in the absence of an L-amino acid donor or a
D-amino acid product, both of the side chains are orientated away
from their respective pockets but are free to move upon ligand
binding.
To understand how D-PhgAT bound its substrates Walton et al
superimposed the active sites of their PLP-free structure with
MesAT with PLP and AKG (PDB: 2YKX), MesAT with PLP:(S)-3-
phenyl--alanine (PDB: 2YKY) and a GSAM AT with PLP:(4S)-4,5-
diaminopentanoate (PDB:2HP2) 48. The Arg54 and Arg412 side
chains of MesAT were proposed to be equivalent residues to the
Arg34 and Arg407 respectively in D-PhgAT. This allowed the
“
missing” Arg34 in the P-pocket to be proposed as the residue that
engages the side chain of the various substrates e.g. the -
carboxylate of L-Glu. Similarly, in the O-pocket Arg407 is thought
to interact with the carboxylate at the C- of amino acids such as
L-Glu.
Now with the Arg34-containing loop observed, we carried out
similar structural comparisons of our PLP-bound structure with
other TA enzymes to gain further insight into the residues involved
in ligand binding in both pockets. We superimposed our PLP-bound
internal aldimine structure with TAs that have structures with useful
bound ligands. Here we used an interesting pair of S-selective -
TAs, Bacillus megaterium (BM-ωTA) and Arthrobacter Ars-ωTA
studied by Dijkstra and colleagues 49. These enzymes share 95%
sequence identity but surprisingly display somewhat different
substrate profiles and structural studies revealed insights into their
mechanism and specificity. The X-ray structure of the PLP:R-MBA
external aldimine bound complex of the BM-ωTA (PDB code:
Figure 2. Crystal Structure of P. stutzeri D-PhgAT. (A)
Functional dimer of the D-PhgAT protein, shown with one
chain as a surface representation and the other as a cartoon
showing the position of the PLP cofactor in stick
representation. (B) Active site pockets of the D-PhgAT protein
showing the PLP internal aldimine and key residues in the
5
G09,) 49 showed the phenyl group of R-MBA binding in the O-
substrate binding pockets. The O- and P-pockets are labelled
following the convention in Wybenga et al 43. The protein
pocket and methyl group in the P-pocket. Dijkstra and colleagues
found that the O-pocket is large enough to accommodate the
phenyl group, in contrast to the smaller P-pocket. The structure also
revealed why this PLP:R-MBA external intermediate was captured
in the unproductive state - the amine of the lysine 298 side chain is
unable to act as a base since it is on the wrong face to remove the
proton from C- By simply inverting the stereochemistry at C- to
the productive S-MBA configuration the enzyme could achieve the
optimal orientation for catalysis. The D-PhgAT also prefers S-MBA
backbone is shown as a cartoon, with key residues shown as
sticks. The two chains in the structure are coloured green and
blue. The final experimental 2mFo-DFc electron density for
the PLP is shown as blue mesh. (C) Cut-away view of the
substrate binding tunnel showing the depth and breadth of the
active site region of the protein. Amino acids and PLP internal
aldimine are shown as stick representations.
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DOI: 10.1039/D0CY01391A
Figure 3. Structural comparison of D-PhgAT with other aminotransferases. (A) Overlay of the P. stutzeri DPhgAT apo (6DVS,
pink sticks) and PLP internal aldimine (6G1F, green and cyans cartoons and sticks) structures with the PLP:R-α-methylbenzylamine
(
R-α-MBA) external aldimine complex of the B. megaterium S-selective aminotransferase (5G09, orange). In the B. megaterium
complex Arg442 swings away from the position of the ring of the R-α-MBA to accommodate this bulky intermediate. The equivalent
residue in the P. stutzeri structure is Arg407, which is found in different conformations in the apo- and PLP-bound structures. (B)
Overlay of the D-PhgAT:PLP internal aldimine with the PLP-external aldimine of Ars ω-TA PLP:L-Ala external aldimine complex
(
5G2Q). In Ars ω-TA PLP:L-Ala structure, the side chain of Arg442 swings in to interact with the C-α carboxylate of the PLP:L-Ala,
this is a 180° flip around the CG of the arginine side chain in comparison to the B. megaterium structure. (C) Schematic representation
of the dual substrate recognition mechanisms of D-PhgAT and the role of Arg34 and Arg407 in the arginine flip/switch model. In the
forward direction the ‘flipping’ Arg407 in the O-pocket is involved in the recognition of the α-carboxylic group of amino donor L-Glu
and can move out of the active site to accommodate the benzyl ring of the product D-Phg. In the P-pocket, the Arg34 side chain is
involved in recognition of the α-carboxylate of the L-Glu (and L-Asp) amino donor.
to R-MBA (Figure S9) suggesting
a
similar discrimination chain of Arg442 is proposed as the arginine switch; swung out in
mechanism.
the PLP-bound complex and swung in to engage with the C-
A comparison of the two structures (Figure 3A) shows how this carboxylate when L-Ala binds. Our overlay of suggests the D-
substrate could be accommodated into the O-pocket; in the BM- PhgAT Arg407 residue plays the equivalent flipping role to Arg442
ωTA: PLP:R-MBA complex the Arg442 is swung out to make way in Ars-ωTA. Our models also suggests that residues His66, His213
for the phenyl ring and Arg407, the equivalent residue in D-PhgAT, from one monomer and Thr303 from the partner monomer play a
is also swung out. Since D-PhgAT prefers L-Ala, L-Glu and L-Asp role in the enantioselective mechanism of the enzyme. These
over their opposite enantiomers we also carried out a comparison residues orientate the ligand to be protonated by Lys269 from the
(
Figure 3B) of the structure of the Arthrobacter Ars-ωTA in complex si-face of the PLP-bound intermediate, generating the D-Phg
with the PLP:L-Ala external aldimine (5G2Q). In Ars-ωTA the side enantiomer (Figures 2B, S15 and S16).
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Journal Name
L-Glu
BZF
View Article Online
DOI: 10.1039/D0CY01391A
-
1
(M-1 s-1)
cat (s-1)
-1 -1
D-PhgAT
K
M
(mM)
k
cat (s )
kcat/K
M
K
M
(mM)
k
M
kcat/K (M s )
WT
26.17 ± 3.63
1.65 ± 0.12
63.21 ± 0.78
3.16 ± 0.46
0.95 ± 0.056
302.80 ± 0.10
R34A*
R407A
H66A
368.33 ± 18.78
133.50 ± 30.5
9.65 ± 0.32
-
0.111 ± 0.0088
0.42 ± 0.012
0.092 ± 0.006
-
0.304 ± 0.004
3.22 ± 0.09
9.76 ± 0.009
-
0.842 ± 0.15
1.51 ± 0.45
11.66 ± 5.95
-
0.079 ± 0.016
0.169 ± 0.012
0.078 ± 0.01
-
93.33 ± 0.037
112.98 ± 0.12
9.57 ± 0.09
-
H213A
Q301A
43.23± 7.90
0.38 ± 0.015
20.59 ± 0.020
9.02± 0.95
0.15 ± 0.012
16.43 ± 0.05
Table 2. Kinetic analysis of the D-PhgAT mutants. Summary of the kinetic Parameters of D-PhgAT WT and mutants for the BZF
and L-Glu substrates. * The reaction couldn´t be saturated as the highest achievable amount in the reaction was 500 mM L-Glu.
We also used this structural analysis to rationalize the L- to D- mutant to allow comparison with the recent results presented by
stereoinversion selectivity of D-PhgAT. Proton transfer of the C-4’ Walton et al 48. The results of this mutagenesis study, summarized
of the resulting PLP:quinonoid intermediate is the key to the in Table 2, show the effect of these changes on the affinity and
enantioselectivity and Jomrit et al, determined that this occurred catalytic efficiency towards both L-Glu and BZF in the forward, D-
from the si face of the intermediate 50. The L-Glu and D-Phg must Phg synthesis direction.
bind in inverted orientations with respect to each other. The As we predicted, the two arginine mutants R34A and R407A
aromatic side chain of D-Phg (and the other aromatic amino acids displayed a much lower affinity towards L-Glu with a K
M
of 368.33
we generated) must therefore exchange for the C- carboxylate of (~14 fold) and 133.50 mM (~5 fold) respectively vs the wild-type K
M
L-Glu (O-pocket) and, similarly, the carboxylate at C- of D-Phg of 26.17 mM. The kcat and the catalytic efficiency is substantially
can swap into the site (P-pocket) that binds the -carboxylate of the disrupted by the mutations for both substrates by approximately
L-Glu side chain. This conformational flexibility requires mobile side 200- and 20-fold respectively. The proposed role of Arg34 as a key
chains in the two pockets and the two arginine residues (R34 and residue in binding L-Glu is strengthened since we found it was not
R407) have the ability to provide such dynamic movement. They possible to saturate the enzyme with the highest achievable
are ideally placed to be able to swing in to engage with carboxylate concentration of L-Glu in the assay (500 mM). Like all AT enzymes,
residues directly in electrostatic interactions. Clearly, the active site the catalytic cycle involves many PLP-derived intermediates
2
must be highly dynamic during catalytic cycles that bind and including the true amine donor PMP which delivers the –NH group
release amino acceptors and products, depending on which to AKG to give D-Phg in that direction. In the absence of PLP-bound
direction it is operating (Schemes 1 and 2). Moreover, we also intermediate structures we used our combined sequence, structural
noted the presence of Q301 in the P-pocket in our structure, and and mutagenesis results to envisage how the substrates and
this adds further weight to its important role in the catalytic products are recognized (Figure 3C). We suggest that Arg407 is
mechanism. The importance of Q301 was identified in a recent cell- the key “flipping” residue that recognizes the C- carboxylate of the
free extract, high-throughput, saturation mutagenesis screen of D- L-Glu amino donor as well as being able to make way for the side
PhgAT catalysing the reverse reaction of D-Phg conversion to L- chains of the aromatic products. Its partner Arg34 is involved in the
Glu 48. This contrasts with our assay which monitors aromatic recognition of the amino donor side chain carboxylic acid group, L-
amino acid production and AKG release. In their screen Walton et Glu is preferred but the shorter L-Asp can also be accepted. This
al focused on three residues in the O-pocket (H66, H213 and R407) arginine ‘switch’ and substrate promiscuity requires a flexible side
and two, in what they predicted, in the absence of defined electron chain and the mobility of Arg407 was revealed by comparing our
density, to be the P-pocket (R34 and Q301). It is interesting to note PLP-bound structure with the two apo-D-PhgAT structures (PDBID:
that, like us, they also found D-PhgAT displayed some substrate 2CY8 and PDBID: 6DVS) 40,50. We found that this sidechain adopts
promiscuity – D-Phg was the best substrate but also showed ~8.6% a different orientation in each structure, suggesting that it is able to
relatively activity with D-Trp and ~82% relative activity with (S-)-4- move to accommodate substrate binding during catalysis (Figure
phenyl-4-aminobutyrate.
4).
We gained further insight into the properties of D-PhgAT by The H213A mutant was totally inactive in all the tested conditions,
carrying out a sequence alignment of the two D-PhgATs from P. suggesting that this residue is crucial to positioning the substrate in
stutzeri and P. putida with well characterized ATs from the same the O-pocket. This is in contrast to the H213A mutant described by
group III fold, but with opposite product enantioselectivity (Figure Walton et al which displays ~45% activity in the reverse direction.
S17). This revealed that the key residues (Arg34, His66, His213, Interestingly, H213N was 150% more active than the wild type but
Gln301 and Arg407) identified in our structural analysis are found a rationale for this dramatic increase is not clear. In our hands both
only in the stereo-inverting enzymes (also referred to as R- the H66A and Q301A mutants retained some activity and by
selective) that produce D-products from L-amino acid donors. This analysis of the structure we suggest that these residues are
suggests that this stereo-inverting property has arisen from using involved in amino-acceptor binding and a hydrogen bonding
the group III TA fold and incorporating these four key amino acids. network surrounding the PLP cofactor respectively (Figure S15).
Since we found these arginine residues in the active site, we carried Similarly, in their screen Walton et al found that mutations at H66
out a targeted mutagenesis study and mutants of these two and Q301 resulted in diminished activity.
residues, R34A and R407A, as well as the two histidine residues,
H66A and H213A, were obtained. We also included the Q301A
8
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DOI: 10.1039/D0CY01391A
Figure 4. Comparison of apo- and PLP-bound forms of D-PhgAT highlights the mobility and movement of Arg407 and loops
around PLP-binding site. Our PLP-bound structure shown in salmon with two apo-forms in orange (PDBID: 2CY8) and blue (PDBID:
6DVS). The disordered, flexible loops, defined only in the PLP-bound structure, are shown as red ribbons with terminal ordered
residues labelled and shown as sticks. For clarity the inset shows a zoom in of the positions of the Arg407 residue in each structure.
Conclusions
that are involved in the catalytic mechanism and potentially control
In conclusion, using the P. stutzeri D-PhgAT we describe a the exquisite R-selectivity of the enzyme. These features suggest
biocatalytic approach to a preparative-scale synthesis of D-Phg in that the O-pocket of D-PhgAT could be engineered to accept even
high % e.e. and yield. The widespread use of PLP-dependent ATs bulkier pro-chiral substrates and convert them to enantiopure R-
for industrial biotechnology has been hampered by the unfavorable products. Our study paves the way for D-PhgAT engineering in
reaction equilibrium towards product formation. This can be order to further expand the substrate scope of this enzyme 54,55. In
overcome by the use of “smart amine donors”, coupled recycling future, structures of the enzyme with substrates and products,
systems or removal of the keto co-product 24,51,52. Unfortunately, the combined with a directed evolution campaign, will allow the product
commonly used iPrA was not accepted by D-PhgAT so here we scope of D-PhgAT to be further expanded to take advantage of the
used a large excess of the inexpensive amino donor L-Glu (~20 unique properties of this versatile biocatalyst.
times K
optimized system gives conversions (93%) that are higher than Experimental
those previously reported 28.
General
M
) to drive the equilibrium towards product formation. Our
A recent report described the engineering of three recombinant E. Standard chemical reagents (D-Phg, D-Hpg, or other amino acids,
coli strains that co-expressed four, seven and nine enzymes, CoASH, buffers) and enzymes were from Sigma, VWR, Alfa Aesar
including the P. stutzeri D-PhgAT as the final step. These whole and Acros Organic.
cell biotransformations produced a range of D-Phg derivatives in
one pot from racemic mandelic acid, styrene and L-Phe starting Cloning and protein expression
materials 53. Since we have shown the D-PhgAT to be a versatile The full-length, codon optimized dpgA gene (Pseudomonas stutzeri
biocatalyst in being able to use a range of amino acceptors it ST-201, UNIPROT code: Q6VY99) was purchased from GenScript
suggests that similar cascades could be constructed to allow and cloned into pET 15b plasmid to give a recombinant D-PhgAT
conversion of simple building blocks to produce a variety of with a non-cleavable N-terminal His6-tag. The construct was used
enantiopure aromatic D-amino acids 53.
to transform E. coli BL21 (DE3) competent cells and selection was
The determination of the first crystal structure of the D-PhgAT with carried out on agar plates containing ampicillin (100 µg/mL;
its bound PLP cofactor has shed light on the unique stereo-inverting LB/Amp100). A single colony was used to inoculate 250 mL of
and enantioselective properties of the enzyme. The broad LB/Amp100 broth and the overnight culture was grown at 37°C with
substrate scope is explained by a large active site cavity and two shaking at 250 rpm. The overnight culture was used to inoculate 1
pockets each containing an essential Arg residue that is crucial for L of fresh LB/Amp100 broth and grown to an A600 of 0.6–0.9. Protein
the catalytic activity. Our study highlights key active site residues expression was induced by addition of IPTG to a final concentration
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Journal Name
of 0.1 mM and growth was continued for 16 h at 20°C. Cells were
harvested by centrifugation (Thermo Scientific Multicentrifuge X3R) High performance liquid chromatography (H DPOL I C: 1 )0 .a1 n0 3a 9l y/ sD i0s CY01391A
View Article Online
at 4000 x g for 30 min at 4°C.
For the HPLC method, reactions containing 0.51 mg/mL D-PhgAT,
-200 mM L-Glu, 0-20 mM amino acceptors
(BZF/HBZF/IPA/PPA/HPA) in 0.1 M CAPS pH 9.5, 150 mM NaCl,
0
Site directed mutagenesis
The D-PhgAT R34A, H66A, H213A, Q301A and R407A mutants 50 M PLP buffer were incubated at 37°C be-fore being terminated
were constructed according to the overlapping primer site-directed at 15 minutes by diluting 40 fold in the chiral mobile phase: 0.025%
mutagenesis (SDM) method with the primer pairs outlined in the triethylammonium acetate (TEAA):MeOH (50:50). Reactions were
supporting information. Colonies were picked and sequenced, and then analysed by chiral HPLC using a Chirobiotic T column (Astec,
based on the results, mutants were taken forward for analysis. The chiral phase Teicoplanin, 5 μm, 250 mm × 4.6 mm) and the
mass of each D-PhgAT mutant was confirmed by LC electrospray following isocratic conditions: mobile phase: 0.025% TEAA:MeOH
−1
ionization mass spectrometry (ESI–MS).
(50:50, v/v), flow rate: 1 mL min , λ: 205 nm, temperature: RT, run
time: 30 min. The column was calibrated with single enantiomers of
the amino acids. The conversion was used to measure enzyme
D-PhgAT purification
All purification steps were carried out at 4°C. Cells were activity. For the detection of non-natural substrates such as D-
resuspended in lysis buffer (0.1 M CAPS pH 9.5, 150 mM NaCl, 20 phenylalanine, D-tyrosine and D-tryptophan the same procedure
mM imidazole, 50 M PLP) with the addition of DNase (0.2 mg per was applied. Lab solution software (Shimadzu) was used to
1
0 mL buffer) and lysed by sonication for 15 cycles (30 sec on, 30 measure the area under the product peaks. To determine the
sec off). The lysed cell suspension was cleared by centrifugation kinetic parameters, reactions were carried out in triplicate and
Thermo Scientific Multicentrifuge X3R) at 14,000 x g for 40 min at quenched after 15 minutes by 1:40 dilution in the chiral mobile
°C. The cell-free extract was loaded onto a 1 mL HisTrap nickel phase. The data were analyzed using the Michaelis–Menten model
(
4
affinity column (GE Healthcare). The column was washed with and a nonlinear regression fit on GraphPad The data points were
binding buffer for 20 column volumes, then the protein eluted with taken as the average values, with the errors calculated as the
an imidazole gradient (10 to 500 mM) over 30 column volumes. standard deviations.
Protein containing fractions were analysed by 12% SDS-PAGE and
concentrated to 1 mL using the Vivaspin 20 MWCO 30000 Amino donor screening
(
Sartorius). The concentrated D-PhgAT was loaded onto a pre- A D-PhgAT aliquot was thawed and incubated on ice with 50 M
equilibrated (0.1 M CAPS, 150 mM NaCl, 50 μM PLP) HiPrep™ PLP for ~2h in order to be sure it was fully loaded with fresh PLP.
6/600 Superdex™ S-200 size exclusion column (120 mL). Reactions containing 0.51 mg/mL D-PhgAT, 10 mM L/D amino
1
Recombinant protein was eluted at a flow rate of 1 mL/min in buffer. donor, 10 mM BZF in 0.1 M CAPS pH 9.5, 150 mM NaCl, 50 M
The purity of the recombinant proteins was analysed by 12% SDS- PLP buffer were incubated at 37°C before being terminated at 18 h
PAGE.
by diluting 40-fold in the chiral mobile phase. Reactions were then
analysed by chiral HPLC using a Chirobiotic-T column as stated
before. Percentage conversions were normalized relative to L-Glu
Spectroscopic analysis
All UV-visible spectra were recorded on a single-beam Cary 50 UV- as the best amino donor.
vis spectrometer and analysed using Origin. To convert the apo-D-
PhgAT to the holo-D-PhgAT form the enzyme was dialyzed for 2 h Biotransformation reactions
at 4°C against 0.1 M CAPS (pH 9.5) containing 150 M NaCl and The large scale biotransformation was performed in a 100 mL
50 μM PLP. Excess PLP was removed by passing the protein conical flask in a final volume of 20 mL. For each component of the
through a PD-10 (Sephadex G- 25M) de-salting column (GE reaction the pH was adjusted to 9.5 before being added to the
Healthcare) before any spectrophotometric measurements were reaction mixture. Freshly purified D-PhgAT (loaded with PLP) was
taken. The concentration of recombinant D-PhgAT was 20 μM and added at a final concentration of 0.2 mg/mL, 0.4 mg/mL or 1mg/mL
the spectrophotometer was blanked with 0.1 M CAPS (pH 9.5) (4 µM, 8 µM, 20 µM) in 0.1 M CAPS buffer pH 9.5, 150 mM NaCl,
containing 150 mM NaCl and 50 μM PLP.
50 M PLP containing 1 g of BZF (~133 mM) and 500 mM L- Glu.
The biotransformation was carried out at 37°C at 120 rpm. A
negative control with all the reagents omitting the enzyme was
ketoglutarate dehydrogenase assay (AKGDH assay)
The D-PhgAT activity was monitored by coupling the reaction with performed at the same time. Time points were collected at time 0,
the ketoglutarate dehydrogenase (AKGDH) since 15 min, 30 min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 24h, 36h. Each
ketoglutarate (AKG) is the product of the first half reaction. The time point was collected in triplicated and processed as described
final volume of the reactions was 250 μl and contained 0.1 M CAPS above. At each time point reactions were terminated by diluting 40

pH 9.5, 150 mM NaCl, 1mM MgCl
2
, 1 mM CaCl
2
, 0.05 mM EDTA, fold in the chiral mobile phase and analysed by HPLC.
+
50 M PLP, 1 mM CoASH, 3 mM NAD , 0.2 U KGDH, 0 to 50 mM
L-Glu, 0 to 20 mM benzylformate (BZF). These reagents were pre- X-ray crystallography.
incubated at 35 °C and the reaction was blanked. The reaction was Purified D-PhgAT, at 9.1 mg/mL, was crystallized by hanging-drop
initiated by adding 1 M D-PhgAT. The increase in absorbance at vapor diffusion at 18°C. Yellow crystals were produced by adding 1
+
3
40 nm resulting from the enzymatic conversion of NAD to NADH L of 9.1 mg/mL D-PhgAT in 0.1 M CAPS (pH 9.5), 150 mM NaCl,
2
after AKG production by the D-PhgAT was monitored over 1h on a 50 M PLP to 1 L of 0.1 M TrisHCl (pH 7.5), 0.2 M MgCl , 10%
BioTek Synergy HT plate reader with Costar 96-well plate. The data (v/v) polyethylene glycol 8000 (PEG 8000). Crystals were cryo-
from the first 15 min were analyzed using the Michaelis–Menten protected with a solution containing 0.1 M TrisHCl (pH 7.5), 0.2 M
model and a nonlinear regression fit on GraphPad gave values of MgCl
2
,
10% (v/v) polypropylene glycol 8000, 20% (v/v)
and kcat (εNADH = 6220 M-1 cm ).
-1
polypropylene glycol 200 PEG and then flash-cooled by immersion
K
M
8
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ARTICLE
in liquid nitrogen. Datasets were collected on beamline I03 at the
Diamond Light Source (Didcot, UK) at 100 K using a Pilatus 6M 10
detector. Diffraction data were integrated and scaled using XDS 46
and symmetry-related reflections were merged with Aimless. The
resolution cut off used for structure determination and refinement
was determined based on the CC1/2 criterion proposed by Karplus
and Diederichs. The structure of D-PhgAT was determined by
molecular replacement using the D-PhgAT incomplete structure
with the PDB code 2CY8. A single solution comprising three dimers
in the asymmetric unit was found using Phaser. The initial model
was rebuilt using Phe-nix.autobuild followed by cycles of
refinement with Phenix.refine and manual rebuilding in Coot. The
final model was refined with automatically determined TLS groups
and isotropic B-factors. The model was validated using MolProbity.
360.
View Article Online
F. Steffen-Munsberg, C. Vickers,DHO.I:K10o.h10ls3,9H/D.0LCaY0n1d3,9H1A.
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Joosten, P. Berglund, M. Höhne and U. T.
Bornscheuer, Biotechnol. Adv., 2015, 33, 566–604.
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11
12
13
2013, 17, 751–759.
Structural superimpositions were calculated using Coot. 14
Crystallographic figures were generated with PyMOL (Schrodinger
LLC). Data collection and refinement statistics are shown in Table 15
S2. X-ray diffraction images are available online at Zendo
P. P. Taylor, D. P. Pantaleone, R. F. Senkpeil and I. G.
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C. K. Saville, J. M. Janey, W. R. Jarvis, J. C. Colbeck,
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000, 8, R1-6.
1
.2r3pre, Schrödinger, LLC). The RMSD values for each overlay
are: 6G1F with 5G2Q – 2.18 Å over 1610 atoms; 6G1F with 5G09
2.12 Å over 1546 atoms; 6G1F with 6DVS – 0.351 Å over 2205
17
18
19
20
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Conflicts of interest
The authors report no conflicts of interest with this work.
Acknowledgements
We thank the Industrial Biotechnology Innovation Centre (IBIOIC) 21
for a PhD studentship (AS) and proof of concept funding (MKA).
We thank the EPSRC CRITICAT Centre for Doctoral Training for 22
the award of a PhD studentship (to SDC, EP/L016419/1).
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Notes and references
The X-ray crystal structure of the D-PhgAT PLP-bound
internal aldimine reported here is available in the Protein Data
Bank (https://www.rcsb.org/) with the accession code 6G1F.
‡
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