Pseudomonas fluorescens Strain R124 Encodes Three Different MIO Enzymes

Article abstract of DOI:10.1002/cbic.201700530

A number of class I lyase-like enzymes, including aromatic ammonia-lyases and aromatic 2,3-aminomutases, contain the electrophilic 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) catalytic moiety. This study reveals that Pseudomonas fluorescens R124 strain isolated from a nutrient-limited cave encodes a histidine ammonia-lyase, a tyrosine/phenylalanine/histidine ammonia-lyase (XAL), and a phenylalanine 2,3-aminomutase (PAM), and demonstrates that an organism under nitrogen-limited conditions can develop novel nitrogen fixation and transformation pathways to enrich the possibility of nitrogen metabolism by gaining a PAM through horizontal gene transfer. The novel MIO enzymes are potential biocatalysts in the synthesis of enantiopure unnatural amino acids. The broad substrate acceptance and high thermal stability of PfXAL indicate that this enzyme is highly suitable for biocatalysis.

Full text of DOI:10.1002/cbic.201700530

DOI: 10.1002/cbic.201700530
Full Papers
Pseudomonas fluorescens Strain R124 Encodes Three
Different MIO Enzymes
Pꢀl Csuka,[a] Vivien Juhꢀsz,[a] Szabolcs Kohꢀri,[b] Alina Filip,[c] Andrea Varga,[c]
Pꢁter Sꢀtorhelyi,[b] Lꢀszlꢂ Csaba Bencze,[c] Hazel Barton,[d] Csaba Paizs,*[c] and
Lꢀszlꢂ Poppe*[a, c, e]
A number of class I lyase-like enzymes, including aromatic am-
monia-lyases and aromatic 2,3-aminomutases, contain the elec-
trophilic 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)
catalytic moiety. This study reveals that Pseudomonas fluores-
cens R124 strain isolated from a nutrient-limited cave encodes
a histidine ammonia-lyase, a tyrosine/phenylalanine/histidine
ammonia-lyase (XAL), and a phenylalanine 2,3-aminomutase
(PAM), and demonstrates that an organism under nitrogen-lim-
ited conditions can develop novel nitrogen fixation and trans-
formation pathways to enrich the possibility of nitrogen me-
tabolism by gaining a PAM through horizontal gene transfer.
The novel MIO enzymes are potential biocatalysts in the syn-
thesis of enantiopure unnatural amino acids. The broad sub-
strate acceptance and high thermal stability of PfXAL indicate
that this enzyme is highly suitable for biocatalysis.
Introduction
Biocatalysis is an intensively developing field of synthetic
chemistry. Enzymes can catalyze stereoselective reactions to
produce valuable intermediates, bulk materials, fine chemicals,
and drugs.[1,2] Enantiopure a- and b-amino acids and their de-
rivatives are frequently used in the pharmaceutical industry.[3]
More and more natural and unnatural amino acids are used to
create novel peptides and cyclopeptides as effective antibiot-
ics.[4]
of aromatic amino acids is difficult and starts non-oxidatively
by ammonia-lyases.[5a] Aromatic amino acid ammonia-lyases
acting on l-histidine, l-phenylalanine, or l-tyrosine as natural
substrates are closely related and share high similarity to aro-
matic 2,3-aminomutases acting on l-phenylalanine or l-tyro-
sine as natural substrates.[6] All members of this enzyme super-
family, involving aromatic ammonia lases and 2,3-aminomutas-
es, contain the unique 3,5-dihydro-5-methylidene-4H-imidazol-
4-one (MIO) catalytic moiety,[5c,7] which forms post-translation-
ally from an XSG triad (X is A in most cases, but may also be T,
S, or C). The autocatalytically formed MIO acts as an electro-
phile and plays an essential role in the catalysis of ammonia
elimination/addition or the 2,3-amino shift from several aro-
matic amino acids.[6]
The enzymes in this study belong to enzyme classes playing
a major role in nitrogen metabolism related to aromatic amino
acids in microbes, fungi, plants, and animals.[5] The metabolism
[a] P. Csuka, V. Juhꢀsz, Prof. Dr. L. Poppe
Department of Organic Chemistry and Technology
Budapest University of Technology and Economics
Mu˝egyetem rkp. 3, 1111 Budapest (Hungary)
E-mail: poppe@mail.bme.hu
Ammonia-lyase activity in nature is required for non-oxida-
tive metabolism of the amino acids by ammonia elimination.
Histidine ammonia-lyase (HAL; EC 4.3.1.3), tyrosine ammonia-
lyase (TAL; EC 4.3.1.23), phenylalanine ammonia-lyase (PAL; EC
4.3.1.24), and phenylalanine/tyrosine ammonia-lyase (PTAL; EC
4.3.1.25) belong to the lyase I like superfamily of enzymes that
catalyze b-elimination reactions and are active as homotetram-
ers. In humans, HAL catalyzes the first step of the degradation
of l-histidine. A deficiency of HAL in humans causes histidine-
mia.[8] In addition, the produced urocanate has been suggested
to function as a sun blocker in human skin.[9] In plants and
fungi, PAL is the first key enzyme in secondary metabolism to
create starting molecules (cinnamate, p-coumarate) for the
phenylpropanoid biosynthesis towards flavonoids, lignins, an-
thocyanins, and stilbenes.[10,11]
[b] S. Kohꢀri, Dr. P. Sꢀtorhelyi
Fermentia Microbiological Ltd
Berlini fflt 47–49, 1049 Budapest (Hungary)
[c] A. Filip, A. Varga, Dr. L. C. Bencze, Prof. Dr. C. Paizs, Prof. Dr. L. Poppe
Biocatalysis and Biotransformation Research Center
Faculty of Chemistry and Chemical Engineering
Babes¸-Bolyai University of Cluj-Napoca
Arany Jꢀnos str. 11, 400028 Cluj-Napoca (Romania)
E-mail: paizs@chem.ubbcluj.ro
[d] Prof. Dr. H. Barton
Department of Biology, The University of Akron
ASEC West Tower 178, Akron, OH 44325 (USA)
[e] Prof. Dr. L. Poppe
SynBiocat Ltd, Szilasliget u. 3
1172 Budapest (Hungary)
Supporting information and the ORCID identification numbers for the
authors of this article can be found under https://doi.org/10.1002/
cbic.201700530.
In addition to their important roles in nature, the MIO en-
zymes—due to their tolerance towards unnatural amino acids
as substrates and the reversibility of their reaction—constitute
This article is part of a Special Issue on Biocatalysis, which commemorates
the BioTrans2017 Symposium in Budapest, Hungary.
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a natural biocatalysis toolbox for syntheses of enantiopure a-
and b-amino acids.[5c,6]
Pseudomonadaceae species, which is one of the most diverse
genera, are found throughout the world.[38] Pseudomonas fluo-
rescens is a very commonly occurring microbe and plays an es-
sential role in agricultural microflorae as biocontrol against
plant diseases in soil media. P. fluorescens strains from diverse
geographic locations have significantly different phenotypic
properties. Some strains of P. fluorescens are able to synthesize
hydrogen cyanide,[39] 2,4-diacetylphloroglucinol,[40] and pyolu-
teorin[41] to protect the roots of plants from fungal infections,
but separate classes cannot synthesize pyoluteorin, only hydro-
gen cyanide and 2,4-diacetylphloroglucinol.[40]
HALs are less similar to PALs, TALs, and PTALs in that they
can act only on a narrow substrate range, just l-4-fluorohisti-
dine,[12] l-4-nitrohistidine,[13] and l-His.[5c,6] Several heteroarylala-
nines and -acrylates could interact with HAL as strong inhibi-
tors, but not as substrates.[14]
PALs exhibit high similarity to TALs and PTALs and accept a
broad range of arylalanines as substrates,[5c,6] such as various
substituted phenylalanines,[15,16] heteroarylalanines,[15b,17] and
even propargylglycine.[18] A single amino acid in the aromatic
binding region of the active site was identified as a critical po-
sition switching between PAL and TAL activities in several
cases. A single mutation of H89F in Rhodobacter sphaeroides
turned a TAL into PAL,[19] whereas the F144H variant of Arabi-
dopsis thaliana PAL exhibited TAL activity.[19a] Several aromatic
ammonia-lyases accepting l-Phe and l-Tyr comparably as sub-
strates are classified as PTAL. PTALs were found in Ascomycota,
Basidiomycota, and Streptophyta species.[20] Due to their broad
substrate scope and reversibility of the ammonia-lyase activity,
PALs and PTALs could be used to synthetize unnatural a-
amino acids, such as arylalanines or heteroarylalanines, under
ammonia-rich conditions.[5c,6]
The P. fluorescens R124 strain was isolated from a nutrient-
limited orthoquartzite sandstone cave.[42] Nutrient limitation
puts environmental pressure on the living organisms to use
novel metabolic pathways. Organisms can adapt to nutrient
limitations by various means, for example, by collection of
novel genes with horizontal gene transfer and by evolving
novel metabolic pathways.[42]
HAL activity from P. fluorescens was identified a long time
ago, first in cell-free extract[43] and later in the purified
enzyme.[44,45]
Although no PAL/TAL activity has been found in P. fluores-
cens until now, p-coumarate formation realized by metabolism
engineering in the closely related Pseudomonas putida indicat-
ed the presence of PTAL activity in a Pseudomonas species.[46]
As part of our efforts to obtain novel MIO enzymes from or-
ganisms living under extreme conditions and locations, herein
we describe the identification and characterization of a HAL, a
XAL, and a PAM from P. fluorescens R124 isolated from an or-
thoquartzite sandstone cave[42] (Scheme 1).
Aromatic b-amino acids play important roles as natural
building blocks in the evolution; growth; and competition of
microbes, fungus and plants.[21] The production of taxol by
Taxus species (T. chinensis, T. cuspidata, T. wallachiana, and
T. canadensis),[22–25] andrimid by Pantoea agglomerans,[26] and
enedyine C-1027 chromophore by Streptomyces globisporus[27]
indicated that an aromatic 2,3-aminomutase in the producer
organism was necessary to form the b-amino acid related frag-
ment in these secondary metabolites from the corresponding
a-amino acid. In addition to their role in nature, phenylalanine
2,3-aminomutases (PAMs) gained increasing synthetic impor-
tance in the production of various unnatural b-arylala-
nines.[24,28–34] PAM catalysis starting from a-amino acids or race-
mic b-amino acids could be applied to produce various un-
natural b-amino acids through kinetic resolution or asymmetric
ammonia addition to arylacrylates, even in cascades coupled
with isomerization of racemic a-amino acids in conjunction
with a promiscuous racemase.[32,34] Furthermore, PAMs were
useful to create enantiopure b-amino acids from arylacrylates
under ammonia-rich conditions.[24,29]
The PAMs ((R)-b-Phe forming: EC 5.4.3.10 and (S)-b-Phe
forming: EC 5.4.3.11) and the tyrosine 2,3-aminomutases
(TAMs, EC 5.4.3.6) constitute the isomerase subfamily of the
MIO enzymes.[6] Due to the preference of various PAMs and
TAMs towards different enantiomers of the produced b-amino
acid, these enzymes are suitable biocatalysts to produce either
desired enantiomer of an aromatic b-amino acid. PAMs of pro-
karyotic origin, such as P. agglomerans,[35,36] are (S)-selective (EC
5.4.3.11), whereas PAMs of eukaryotic origin, such as Taxus spe-
cies,[24,25] are (R)-selective (EC 5.4.3.10). Although all TAMs are
classified under one EC entry (EC 5.4.3.6), there are two forms
of TAMs with opposite enantiopreference. TAM from Chondro-
myces crocatus can produce (R)-b-tyrosine from l-Tyr,[37] where-
as TAM from S. globisporus can give access to (S)-b-tyrosine.[16]
Scheme 1. The reactions catalyzed by the three MIO enzymes from P. fluores-
cens R124: PfHAL: ammonia elimination from l-His; PfXAL: ammonia elimi-
nation from l-Phe, l-Tyr, or l-His; PfPAM: 2,3-amino shift between (S)-a- and
(S)-b-phenylalanine.
Results and Discussion
All three novel MIO enzymes from P. fluorescens R124 were
identified by bioinformatics tools and cloned from genomic
DNA. The thermotolerance of the novel MIO enzymes was
characterized by their unfolding temperature and temperature-
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dependent biocatalytic activity. The biocatalytic properties of
the new enzymes were characterized by their ability to accept
natural aromatic amino acids as substrates, by their kinetic pa-
rameters, optimum pH and temperature of their reaction with
natural substrates, and the effect of divalent metal ions on the
enzyme activity.
trogen-limited conditions in the sandstone-rich cave through
the development of novel nitrogen fixation and transformation
pathways to enrich the possibility of nitrogen metabolism.[42]
Multiple sequence alignment showed that ASG triads in
PfHAL and PfXAL, but a TSG triad in PfPAM, were the precursor
of the MIO catalytic group (Figure 1, motif from position 141).
The MIO created from the TSG triad and the sequence motif
for the aromatic binding pocket (Figure 1, from position 76) of
PfPAM was similar to other (S)-selective bacterial PAMs (e.g.,
PaPAM[25]). The phenylalanine residue of the DFQD sequence
motif (Figure 1, from position 413) in PfPAM sequence played
an important role in shifting the ammonia-lyase activity of
PaPAM towards the main 2,3-aminomutase activity.[36] The
PfPAM sequence from P. fluorescens R124 as a candidate (S)-
PAM was already hypothesized without characterization.[33]
Analysis of genomes of further P. fluorescens strains revealed
that PfPAM identified in the R124 strain was unique and could
not be identified in genomes of other P. fluorescens strains. Be-
cause HAL activity was already identified in P. fluorescens,[43–45]
it was not surprising that an SH dyad characteristic of HAL ac-
tivity was found in the aromatic binding region of PfHAL (iden-
tical to PpHAL in positions 83–84). Moreover, PfHAL from strain
R124 exhibited 84% identity to PpHAL (UniProtKB: P21310). In
the PfXAL sequence, the motif characteristic for the lyase to
mutase activity shift (ANQD, see positions 413–417 in Figure 1)
proved to be identical to that found in RsTAL. This was in
good agreement with the finding that PfXAL accepted l-Tyr as
the best substrate. Analysis of the sequence motif of the aro-
matic group binding site in PfXAL revealed a unique FH dyad
corresponding to positions 83–84 of PpHAL. Corresponding to
this position, an FL dyad is present in the aromatic binding
region of many PALs (see AvPAL in Figure 1). On the other
hand, a HL dyad corresponding to positions 83–84 of PpHAL is
common for many TALs (see RsTAL in Figure 1). Thus, partial
similarity of the FH dyad PfXAL to all of the dyads within the
aromatic binding pocket of PALs, TALs, and HALs could ration-
alize the promiscuity of this unique aromatic ammonia-lyase.
Although purified PfHAL was characterized partially,[44,45]
most biochemical data was disclosed for the closely related
HAL from P. putida (PpHAL). PpHAL was one of the most inten-
sively investigated MIO enzymes[5c] with remarkable thermal
properties. The crystal structure of recombinant PpHAL pro-
duced in Escherichia coli revealed the homotetrameric structure
of the enzyme and allowed, for the first time, identification of
the unique electrophilic MIO catalytic moiety.[47,48] PpHAL
proved to be a thermostable enzyme purified by heat induc-
tion from the cell lysate with detectable activity even at
1.58C.[49] PpHAL was activated by zinc ions, but other bivalent
metal ions decreased its activity.[50] The catalytic importance of
various key residues in the active site of PpHAL were demon-
strated by site-directed mutations and kinetic studies.[51] Later,
computations confirmed the necessity of the zinc ion for bind-
ing l-histidine to the active site of PpHAL.[52]
Identification of novel MIO enzymes from P. fluorescens
R124
A sequence similarity search by using the BLASTp tool to mine
sequence information from bacterial genome, nucleotide, and
protein sequence databases (NCBI Genome, Nucleotide and
Protein, respectively)[53] enabled us to find novel MIO en-
zymes.[54] To identify novel PALs and PAMs from bacteria living
under extreme conditions, BLASTp searches were performed
by using a PAM from P. agglomerans (PaPAM or AdmH, Uni-
ProtKB: Q84FL5)[25] and a PAL from Photorhabdus luminescens
(UniProtKB: Q7N4T3)[55] as query sequences. Multiple sequence
alignment of the sequence domains characteristic of MIO en-
zymes led to the identification of three novel MIO enzymes in
P. fluorescens R124 strain (denoted as PfHAL, PfXAL, and PfPAM
in Figure 1). This is a noteworthy result because, to the best of
our knowledge, there are no reports on any other organism
encoding three different MIO enzymes. Although the roles of
these MIO enzymes in P. fluorescens R124 are not fully clear, it
could be hypothesized that collection of novel genes by hori-
zontal gene transfer could contribute to adaptation to the ni-
Substrate specificity and kinetics of the MIO enzymes from
P. fluorescens R124
After expressing the three novel MIO enzymes from the P. fluo-
rescens R124 strain, their catalytic ability in ammonia elimina-
tion was tested with all of the natural aromatic amino acids (l-
Figure 1. Sequence codes (UniProtKB) and characteristic sequence domains of the three MIO enzymes from P. fluorescens R124 (PfHAL, PfXAL, and PfPAM)
compared with HAL from P. putida (PpHAL), PAL from Anabaena variabilis (AvPAL), TAL from R. sphaeroides (RsTAL), and PAM from P. agglomerans (PaPAM).
Color codes: red: tyrosine base; blue: key amino acids in the aromatic binding pocket; yellow: amino acids forming the MIO group; orange: amino acids sta-
bilizing MIO; green: arginine binding carboxylic group of the substrate; purple: phenylalanine important for 2,3-aminomutase activity. Numbering corre-
sponds to PpHAL.
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His, l-Phe, l-Tyr, and l-Trp; see Tables S3 and S4 in the Sup-
porting Information). Unsurprisingly, PfHAL could catalyze am-
monia elimination specifically from l-His. None of the MIO en-
zymes from the P. fluorescens R124 strain exhibited detectable
reaction with l-Trp.
PfXAL kinetic behavior was studied with the three aromatic
acids accepted as substrates (l-Tyr, l-Phe, and l-His; Table 1).
PfXAL with l-Tyr exhibited either KM (21 mm) or kcat (0.52 sÀ1);
these values are similar to those found for other bacterial TALs
from Rhodobacter capsulatus (KM =16 mm, kcat =27.7 sÀ1)[56] or
R. sphaeroides (KM =0.1 mm, kcat =0.9 sÀ1).[57] PfXAL with l-Phe
reacted faster (kcat =2.38 sÀ1), but displayed lower affinity (KM =
2.6 mm) than that with l-Tyr. This behavior resembled that of
other bacterial TALs from R. capsulatus (kcat =15.1 sÀ1 and KM =
1.28 mm with l-Phe)[56] or R. sphaeroides (kcat =0.6 sÀ1 and KM =
2.7 mm with l-Phe).[57] PfXAL with l-Phe exhibited a similar
turnover number, but lower affinity than those with AvPAL
(kcat =4.3 sÀ1 KM =60 mm).[58] PfXAL with l-His exhibited measur-
able activity (Usp =9.6ꢄ10À3 UmgÀ1), but did not followed Mi-
chaelis–Menten kinetics. So far, no PAL, TAL, or PTAL has been
reported with measurable HAL activity.
PfXAL proved to be active not just with l-Phe and l-Tyr (sim-
ilar to PTALs classified as a separate enzyme family; EC
4.3.1.25), but also catalyzed ammonia elimination from l-His
(Table S4). This result was in accordance with sequence analy-
sis; this indicated partial similarity of the aromatic binding
domain of PfXAL to TALs, PALs, and HALs (Figure 1). To the
best of our knowledge, PfXAL is the first known aromatic am-
monia-lyase that can catalyze ammonia elimination from three
natural aromatic amino acids (l-Tyr, l-Phe, and l-His). PfXAL
was also tested with dl-Phe to study the ability of the enzyme
as a biocatalyst for kinetic resolution of the ammonia elimina-
tion reaction. The significant decrease in activity of PfXAL-cata-
lyzed ammonia elimination with dl-Phe to 67 and 85% (at 40
and 20 mm concentrations, respectively), relative to that with
l-Phe (at 20 and 10 mm concentrations, respectively), indicated
the inhibitory nature of d-Phe if present in equal amounts rela-
tive to that of l-Phe in dl-Phe (Table S4).
Optimum pH of ammonia-lyases from P. fluorescens R124
The effect of pH on the ammonia-lyase activity of PfHAL with
l-His and PfXAL with l-Phe was investigated between pH 7
and 11 (Figure 2). PfHAL exhibited maximal enzyme activity at
PfPAM exhibited 51% sequence identity to PaPAM, and the
catalytically important motifs of PfPAM, which involved the cat-
alytic tyrosine, the catalytic MIO group, and the phenylalanine-
enhancing aminomutase activity, were identical to those in
PaPAM (see motifs from 54, 140, 192, 279, and 413 in Figure 1).
Consequently, we expected that PfPAM had similar catalytic
properties to those of PaPAM, which performed isomerization
of l-Phe to (S)-b-phenylalanine and exhibited ammonia-lyase
activity as well.[35,36] Unsurprisingly, PaPAM also exhibited 2,3-
aminomutase activity for preferential interconversion of l-Phe
into (S)-b-phenylalanine and significant ammonia-lyase activity
(see Section 9, Table S10, and Figures S22 and S23).
Figure 2. Effect of pH on the ammonia elimination reaction of PfHAL from l-
~
&
*
^
His ( ), PfXAL from l-Phe ( ), PfXAL from l-Tyr ( ), and PfXAL from l-His ( ).
Kinetic constants for the two ammonia-lyases were deter-
mined with all natural aromatic amino acids accepted as sub-
strates (Table 1). PfHAL with l-His exhibited a Michaelis con-
stant (KM) of 2.7 mm and turnover number (kcat) of 65 sÀ1
(Table 1). Kinetic constants of PfHAL were in the same order of
pH 8.5 (Figure 2), which was in good agreement with previous
data on pH profiles of HALs from Pseudomonas sp. with 50%
maximal activity at pH 8.1 and 9.9, and a maximum at around
pH 9.[59] PfXAL exhibited quite low activity with l-Phe, l-Tyr, or
l-His at pH 7 and became more active in the range of pH 8.5–
10. The pH optima of PfXAL were between pH 9 and 10 with
magnitude as those determined previously for PpHAL (kcat
86 sÀ1 and KM =3.9 mm).[51]
=
Table 1. Kinetic parameters of PfHAL and PfXAL compared with other MIO enzymes in ammonia elimination from the indicated substrates at 308C.
Enzyme
Substrate
KM [mm]
2700Æ110
kcat [sÀ1
]
kcat/KM [sÀ1 mmÀ1
]
Usp [UmgÀ1
]
Ref.
[a]
PfHAL
PpHAL
PfXAL
RcTAL
RsTAL
PfXAL
RcTAL
RsTAL
AvPAL
PfXAL[b]
l-His
l-His
l-Tyr
l-Tyr
l-Tyr
l-Phe
l-Phe
l-Phe
l-Phe
l-His
65
86
24.0
22.0
25.0
1.73
9.0
0.91
11.79
0.22
71.7
n.d.
17.2
n.d.
0.13
n.d.
n.d.
0.60
n.d.
n.d.
n.d.
[46]
[a]
3900
21Æ2
0.52
27.7
0.9
2.38
15.1
0.6
[56]
[57]
[a]
16000
100
2600Æ110
1280
2700
[56]
[57]
[58]
[a]
60
4.3
n.d.
>4ꢄ105[b]
9.6ꢄ10À3[c]
[a] This study. [b] PfXAL with l-His did not follow Michaelis–Menten kinetics up to 150 mm l-His. The KM value estimated with the Michaelis–Menten kinet-
ics equation was higher than that indicated. [c] The Usp value for PfXAL with l-His was determined at a 150 mm substrate concentration by using the
actual rate as Vmax
.
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all three substrates. These pH profiles resemble those of most
PALs and PTALs, with pH optima in the range of 8.5–9.[5,6] Con-
sequently, PfHAL and PfXAL exhibited similar pH profiles to
those of most corresponding MIO enzymes.
Effect of divalent cations on the activity of PfHAL and PfXAL
Previous studies with PpHAL revealed that various divalent cat-
ions (Co2+, Cu2+, Mg2+, Ni2+, Zn2+) may alter the enzyme ac-
tivity by influencing the substrate–enzyme interaction.[47]
PpHAL expressed without His-tag was significantly activated by
Zn2+ ions, whereas other cations inhibited the formation of
the urocanate. An investigation into the effect of five divalent
cations on the activity of PfHAL revealed no significant differ-
ence in activity from that of the ethylenediaminetetraacetic
Figure 4. Thermal stability of three MIO enzymes from P. fluorescens R124
(c: PfHAL, c: PfXAL, c: PfPAM) by nanoDSF. dA330 is the first deriva-
tive of the absorbance at l=330 nm.
modulation. Thermal stability of the three MIO enzymes was
characterized by nano-differential scanning fluorimetry DSF
(nanoDSF) measurements (Figure 4).
acid (EDTA)-free control assay in the presence of Zn2+, Mg2+
,
To characterize the enzymes, unfolding events of the pro-
teins were measured with a nanoDSF device by monitoring
the intrinsic tryptophan fluorescence at emission wavelengths
of l=330 and 350 nm. Because the melting temperature (Tm)
of a lipase determined by differential scanning calorimetry
(DSC)[64] was identical to that obtained by nanoDSF,[65] this
technique proved to be a straightforward, simple, and highly
correlative method to traditional techniques.
or EDTA (Figure 3). A slight to moderate decrease in PfHAL ac-
tivity was observed if Co2+, Ni2+, and Cu2+ were present in the
assay. Apart from biochemical evidence,[47] computational stud-
ies[51] also showed that Zn2+ played an important role in HAL
catalysis.
The high thermotolerance of PfHAL was indicated by the
lack of unfolding events observed up to 858C by means of
nanoDSF. This good thermostability of PfHAL was expected be-
cause the isolation process of highly similar PpHAL (expressed
from E. coli) started with heat shock at 708C and resulted in a
catalytically active enzyme after the heat-shock step.[51] PfXAL
also exhibited promising thermal properties with an unfolding
temperature above 808C; this indicates its possible use as a
biocatalyst at elevated temperatures. Although PfPAM showed
the lowest thermal stability among the three MIO enzymes of
P. fluorescens R124, this protein only displayed heat-dependent
structural change above 758C, as determined by nanoDSF.
Next, the long-term thermostability of PfHAL and PfXAL was
tested by incubation of the enzymes for different durations at
different temperatures followed by nanoDSF and activity mea-
surements.
Figure 3. Effect of the presence of divalent cations of the indicated metals
&
&
in l-His elimination by PfHAL ( ) and the l-Phe elimination by PfXAL ( ).
Metal-ion assay media contained EDTA (42.5 mm) and divalent metal ions
(55.5 mm). The reactions were performed in triplicate.
PfXAL activity with l-Phe was inhibited strongly by Zn2+
,
less significantly with Co2+ and Cu2+, and only weakly by Ni2+
and Mg2+ (Figure 3), relative to the EDTA-free and EDTA-sup-
plemented controls. This behavior of PfXAL resembled the in-
hibition patterns of divalent metal ions for PTAL from Rhodo-
torula glutinis,[60] PALs from sunflower (Helianthus annuus L.) hy-
pocotyls,[61] and leaf mustard (Brassica juncea);[62] these showed
significant inhibition with Co2+ and Cu2+. Mustard PAL was
fully inhibited by Zn2+ at a concentration of 1 mm.[62] PfXAL
showed no significant inhibition or activation in ammonia
elimination from l-His in the presence of 0–300 mm Zn2+ (Fig-
ure S17).
Although the unfolding temperature of PfHAL did not
change after 6 and 24 h incubation between 30 and 608C, the
activity showed significant alterations, depending on incuba-
tion time. Enzyme activity after 24 h incubation at elevated
temperatures between 30 and 608C was significantly lower
than that after 6 h incubation (Figures S19 and S20). This loss
of enzyme activity without alteration in unfolding temperature
can be rationalized by assuming that small local conformation-
al changes within the active site were responsible for inactiva-
tion. Incubation temperatures above 608C caused even further
decrease of the HAL activity.
Thermal properties of MIO enzymes from P. fluorescens R124
Thermal stability is of prime importance from the viewpoint of
the applicability of biocatalysts in industrial processes. The
thermal stability of PALs may contribute to higher productivity
and longer operational stability in biotransformations. More-
over, bacterial PAMs exhibit temperature-modulated ammonia-
lyase activity at elevated temperature;[63] therefore, higher ther-
mal stability of PAMs can provide a wider range of functional
The unfolding temperature dependence of PfXAL between
30 and 608C was similar to that of PfHAL, which showed no
significant alterations as a function of incubation time and
temperature. PfXAL exhibited no significant alteration in the
residual PAL activity tested with l-Phe after various incubation
times and temperatures between 30 and 608C (Figure S18 and
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Table S9). Incubation temperatures above 608C, however, re-
sulted in a decrease of the PAL activity.
Aided by its reverse ammonia-lyase activity, PfPAM proved
to be an applicable biocatalyst to convert (E)-cinnamic acid in
4m ammonium carbamate buffer into a mixture of (S)-Phe
(25%) and (S)-b-Phe (33%, see Table S10). This indicated that
PfPAM might be applied as an enzyme in asymmetric synthe-
ses of (S)-b-amino acids starting from an easy-to-produce achi-
ral substrate.
Finally, the temperature dependence of the ammonia-lyase
activity of PfHAL and PfXAL was examined in the range of 30–
608C (Figure 5). PfHAL showed a temperature optimum at
Conclusion
A HAL, XAL, and PAM were identified in the genome of P. fluo-
rescens R124 strain. This strain isolated from a nutrient-limited
cave demonstrated that an organism could utilize more than
two different MIO enzymes to enrich the nutrient utilization
pathways under metabolic stress. The unique substrate accept-
ance and high thermal stability of PfXAL indicated the high po-
tential of this enzyme as a biocatalyst to produce either de-
sired enantiomer of an aromatic amino acid in high enantio-
purity. PfPAM belonging to the class of S-selective PAMs (EC
5.4.3.11) exhibited potential for application as a biocatalyst in
the production of enantiopure aromatic b-amino acids from a-
arylalanines or even from (E)-arylacrylates.
*
Figure 5. Dependence of ammonia-lyase activity of PfHAL (with l-His,
)
*
and PfXAL (with l-Phe, ) on temperature.
around only 408C. This was in accordance with the tempera-
ture-dependent incubation experiments, which indicated high
global structural stability (unfolding temperature), but signifi-
cant inactivation at lower temperatures without global struc-
tural changes. The ammonia-lyase activity of PfXAL with l-Phe
increased continuously upon increasing the temperature from
30 to 608C, in accordance with temperature-dependent incu-
bation experiments. This renders PfXAL as a promising thermo-
tolerant biocatalyst for future biotransformations.
Experimental Section
Materials and methods: Genomic DNA of P. fluorescens R124 was
isolated by Barton and co-workers.[42] Details on further materials
and methods are given in the Supporting Information.
Identification of the genes of MIO enzymes from P. fluorescens
R124: Protein BLAST searches were used to find novel bacterial
PAMs in the NCBI Protein database by using the sequence of PAM
from P. agglomerans (PaPAM, gene ID: AdmH, UniProtKB ID:
Q84FL5) as a query (for details, see Section 2 in the Supporting In-
formation).
PfPAM-catalyzed reactions
Due to the 51% overall sequence identity of PfPAM to PaPAM,
and to the full identity of the conserved catalytically important
motifs (see motifs from 54, 140, 192, 279, and 413 in Figure 1),
it was expected that the two enzymes had similar properties.
The activity and stereoselectivity of the isomerization and am-
monia-lyase reactions catalyzed by PfPAM were tested from
both directions (see Section 10 in the Supporting Information).
PfPAM showed similar stereoselectivity to that of PaPAM[35,36]
and catalyzed the formation of (S)-b-Phe in up to 99% ee from
(S)- or (R,S)-Phe. PfPAM catalyzed the reaction of (R,S)-b-Phe in
the reverse direction and resulted in the formation of (S)-Phe
with 11.8% conversion from the consumed (S)-b-Phe. In addi-
tion to the main aminomutase activity, PfPAM also exhibited
ammonia-lyase activity. Analysis of PfPAM-catalyzed biotrans-
formation from (S)-Phe (2 mm) revealed, after 24 h (in equilibri-
um), the formation of (S)-b-Phe (55%), (S)-Phe (25%), and (E)-
cinnamic acid (20%). A similar reaction from racemic substrate
((R,S)-Phe, 4 mm) resulted in the formation of significantly less
(S)-b-Phe (28%) and (E)-cinnamic acid (4%), along with residual
substrate (68%) enriched in (R)-Phe. A comparison of the reac-
tions starting from (S)- and (R,S)-Phe revealed that (R)-Phe had
an inhibitory effect on the aminomutase and ammonia-lyase
activities. Analysis of the time course of PfPAM bioconversions
showed that the rate of isomerization from (S)-Phe to (S)-b-Phe
proceeded at a higher rate than that of the reverse reaction.
Data also showed that the (S)-Phe/(S)-b-Phe equilibrium was
shifted significantly towards (S)-b-Phe (Table S10).
Cloning of the P. fluorescens R124 MIO enzyme genes: Genomic
DNA of P. fluorescens R124 was applied to clone and multiply the
genes (pfhal, pfxal, pfpam) with Phusion Flash high-fidelity PCR,
which were separated by agarose gel electrophoresis. The forward
and reverse primers contained the NdeI and BamHI restriction en-
donuclease cleavage sites, respectively. The PCR products were ex-
tracted with an EZ-10 spin column. The extracted genes were ligat-
ed in pJET1.2/blunt cloning vector at 228C. The plasmids were
transformed into E. coli XL1 Blue cells. Carbenicilline (Car) and tet-
racycline (TC) were used for positive selection. The genes and
pET19-b expression vector were digested on 378C with NdeI and
BamHI restriction enzymes, followed by separation by agarose gel
electrophoresis. The purified genes and vector DNA were ligated at
228C with T4 DNA ligase. The resulting expression plasmids
(Table 2) were transformed into E. coli XL1 Blue cells and selected
with Car and TC. Finally, the plasmids were transformed into E. coli
Rosetta strain with Car and chloramphenicol (CL) as antibiotics (for
further details of the cloning procedures, see Section 3 in the Sup-
porting Information).
Expression of the P. fluorescens R124 MIO enzymes: Briefly, over-
night cultures of the transformed E. coli Rosetta (DE3)pLysS cells
grown at 378C (10 mL) were used to inoculate lysogeny broth (LB)
in 2 L Erlenmeyer flasks containing sterilized LB medium (500 mL)
and cultures were shaken at 200 rpm at 378C until OD600 =0.6–0.7
(2.5 h), followed by induction with isopropyl b-d-1-thiogalactopyra-
&
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Kinetic constants were determined for PfHAL with l-His (0.1–
30 mm), and for PfXAL with l-Phe (0.1–30 mm), l-Tyr (0.001–3 mm),
and l-His (5–150 mm), as described in Section 9.2 in the Support-
ing Information.
Table 2. Plasmid constructs for the expression of PfHAL, PfXAL, and
PfPAM.
Plasmid
Gene
Restriction cleavage site
Vector
pSBHALPf01w[a]
pSBXALPf01w[a]
pSBPAMPf01w[a]
pfhal
pfxal
pfpam
NdeI and BamHI
NdeI and BamHI
NdeI and BamHI
pET19-b
pET19-b
pET19-b
The metal-ion dependence of PfHAL with l-His and PfXAL with l-
Phe was measured with 42.5 mm EDTA and 55.5 mm metal ions
(Zn2+, Mg2+, Co2+, Cu2+, Ni2+).
[a] Plasmids were designed to express the target enzymes with N-termi-
nal His10 tag.
Analysis of the PfPAM-catalyzed reactions: Biotransformations
with PfPAM (50 mgmLÀ1) contained the substrate (2 mm l-Phe,
4 mm dl-a-Phe, 4 mm dl-b-Phe, and 2 mm (E)-cinnamic acid) in
the following buffers: 100 mm Tris, pH 8.5, for the aminomutase re-
actions; and 4m ammonium carbamate, pH 9.0, for the ammonia
addition reactions (for further details, see Section 10 in the Sup-
porting Information). The enantiomeric composition of the a-Phe
and b-Phe fractions after 18 h reactions with PfPAM was tested on
a ZWIX column (Figures S21–S23). Interconversion between a-Phe
and b-Phe, and conversion to and from (E)-cinnamic acid, were an-
alyzed on a Phenomenex Gemini NX-C-18 column (Table S10 and
Figure S24).
noside (IPTG; 60 mm) and further incubation at 288C, 200 rpm, for
6 h (for further details of protein expression, see Section 4 in the
Supporting Information).
Purification of the P. fluorescens R124 MIO enzymes: Cells were
harvested at 48C by centrifugation (20 min, 4500g) followed by
disruption in lysis buffer (50 mL; Tris, 50 mm; pH 8, 300 mm NaCl,
0.5 mm EDTA; containing lysozyme, RNAse A, and EDTA-free pro-
tease inhibitor cocktail) by sonication on ice for 20 min. After cen-
trifugation (12500 rpm, 20000g, 20 min) the lysate was applied
onto a Ni-NTA agarose affinity column and washed with LS buffer
(50 mm HEPES and 30 mm KCl pH 7.5), HS buffer (50 mm HEPES
and 300 mm KCl pH 7.5), LS buffer again, and then Lim (20 mm imi-
dazole, 50 mm HEPES, 30 mm KCl, pH 7.5) buffer. The target
enzyme was eluted in Him (300 mm imidazole, 50 mm HEPES,
30 mm KCl, pH 7.5) buffer and desalted on a HiTrap column (for
imidazole removal) and finally concentrated by ultracentrifugation
with Amicon filters (for further details of the protein purification,
see Section 5 in the Supporting Information). The native molecular
size of the enzyme was analyzed on a Superdex 200 gel filtration
column (Figures S9–S11). Purity of the target enzyme was checked
by SDS-PAGE analysis (Section 6 and Figure S12 in the Supporting
Information). Protein concentration in the final enzyme prepara-
tions was determined by means of the Bradford method
(3.2 mgmLÀ1 for PfHAL, 4.0 mgmLÀ1 for PfXAL, and 1.7 mgmLÀ1
for PfPAM).
Acknowledgements
We thank support from the New SzØchenyi Development Plan of
Hungary (Development of quality-oriented and harmonized R+
D+I strategy and functional model at BME, TMOP-4.2.1/B-09/1/
KMR-2010-0002). Financial support for project NEMSyB (ID P37_
273, Cod MySMIS 103413; funded by the National Authority for
Scientific Research and Innovation (ANCSI) and co-funded by the
European Regional Development Fund, Competitiveness Opera-
tional Program 2014–2020 (POC), Priority axis 1, Action 1.1; Ro-
mania) and for project PROMYS (grant no. IZ11Z0_166543) from
the Swiss National Science Foundation (SNSF) is gratefully ac-
knowledged. L.P. and C.P. thank support from the EU COST Action
CM1303 (SysBiocat).
Thermal characterization of the MIO enzymes from P. fluorescens
R124: Thermal stabilities of the enzymes were determined by
nanoDSF (at 0.1 mgmLÀ1 of PfHAL, 0.4 mgmLÀ1 of PfXAL and
0.4 mgmLÀ1 of PfPAM concentrations) by using Prometheus NT.48
equipment (NanoTemper Technologies; for details of the measure-
ments, see Sections 1 and 8 in the Supporting Information).
Conflict of Interest
The authors declare no conflict of interest.
Keywords: biocatalysis · gene transfer · lyases · nitrogen ·
phenylalanine
Ammonia-lyase activity assays and kinetic characterization of
PfHAL and PfXAL: Ammonia-lyase activity was assayed by UV ab-
sorbance measurements of the forming arylacrylic acid as a func-
tion of time at 308C for 5 min (4.2 mgmLÀ1 of PfHAL with l-Phe,
30 mgmLÀ1 of PfXAL with l-Phe, and 20 mgmLÀ1 of PfXAL l-Tyr).
Before the assays, the enzyme was preincubated for 5 min at 308C.
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Manuscript received: October 3, 2017
Accepted manuscript online: November 28, 2017
Version of record online: && &&, 0000
&
ChemBioChem 2018, 19, 1 – 9
www.chembiochem.org
8
ꢃ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
The first MIO trio: The P. fluorescens
R124 strain encodes a histidine ammo-
nia-lyase (HAL), a tyrosine/phenylala-
nine/histidine ammonia-lyase (XAL), and
a phenylalanine 2,3-aminomutase (PAM)
to demonstrate that an organism under
metabolic stress can utilize more than
two different 3,5-dihydro-5-methyli-
dene-4H-imidazol-4-one (MIO) enzymes,
which are highly suitable biocatalysts
for the production of enantiopure aro-
matic amino acids.
P. Csuka, V. Juhꢀsz, S. Kohꢀri, A. Filip,
A. Varga, P. Sꢀtorhelyi, L. C. Bencze,
H. Barton, C. Paizs,* L. Poppe*
&& – &&
Pseudomonas fluorescens Strain R124
Encodes Three Different MIO Enzymes
ChemBioChem 2018, 19, 1 – 9
www.chembiochem.org
9
ꢃ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ