Characterization of the active site of histidine ammonia-lyase from
Pseudomonas putida
Dagmar Ro¨ ther
1
,La
´
szlo
´
Poppe
2
, Sandra Viergutz
1
, Birgid Langer
1
and Ja
´
nos Re
´
tey
1
1
Institute for Organic Chemistry, University of Karlsruhe, Germany;
2
Institute for Organic Chemistry, Budapest University of Technology
and Economics, Hungary
Elucidation of the 3D structure of histidine ammonia-lyase
(HAL, EC 4.3.1.3) from Pseudomonas putida by X-ray
crystallography revealed that the electrophilic prosthetic
group at the active site is 3,5-dihydro-5-methylidene-4H-i-
midazol-4-one (MIO) [Schwede, T.F., Re
´

5-methylidene-4H-imidazol-4-one; MIO; site-directed
mutagenesis.
Histidine ammonia-lyase (HAL, EC 4.3.1.3) is the first
enzyme in the nonoxidative degradation pathway of
L-histidine. The enzymic catalysis begins with a Friedel–
Crafts-type reaction, which helps to transform
L-histidine to
trans-urocanate (reviewed in [1]). An analogous mechanism
was proposed for the reaction catalysed by the homologous
enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5)
which converts
L-phenylalanine into trans-cinnamic acid, a
precursor of a great variety of phenylpropanoids [2].
Approximately 30 years ago it was postulated that a
dehydroalanine residue at the active site of both enzymes
acted as electrophilic prosthetic group [3–5]. Mutagenesis
experiments showed that this dehydroalanine is post-
translationally formed from serines 143 and 202 of HAL
and PAL, respectively [6,7]. More recently, the X-ray
structure of HAL was solved at 2.1 A
˚
resolution [8]. It was
shown that the prosthetic group is not dehydroalanine but a
3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO). It
was proposed that this group is formed by cyclization of
an intramolecular 142ASG144 tripeptide followed by
subsequent elimination of two molecules of water (Fig. 1).
A similar mechanism was proposed for the formation of
the p-hydroxy-benzylidene-imidazol-5-one fluorophore
of the green fluorescent protein from Aequorea victoria

(Received 28 June 2001, accepted 5 September 2001)
Abbreviations: HAL, histidine ammonia-lyase; PAL, phenylalanine
ammonia-lyase; MIO, 3,5-dihydro-5-methylidene-4H-imidazol-4-one.
Eur. J. Biochem. 268, 6011–6019 (2001) q FEBS 2001
Site-directed mutagenesis
Mutagenesis was carried out in a C273A mutated gene for
HAL from Pseudomonas putida to permit a subsequent
crystallization without forming polymeric forms of enzyme
[13].
HAL mutants R283I, R283K, H83L, N195A, E414A,
E414Q, Q277A and F329A were performed following
the QuickChange
TM
site-directed mutagenesis system
(Stratagene) [14].
The oligonucleotides used in the mutagenesis reactions
were:
HAL-R283I(1): 5
0
-CGTACTCGCTGATCTGCCAGCCG-
3
0
; HAL-R283I( –): 5
0
-CGGCTGGCAGATCAGCGAGTA
CG-3
0
; HAL-R283K(1): 5
0
-CGTACTCGCTGAAATGC

-GCCAA
CCAGG
CAGACCACGTATCG-3
0
; HAL-E414A(–): 5
0
-
CGATACGTGGTCT
GCCTGGTTGGC-3
0
HAL-E414Q(1): 5
0
-GCCAACCAGCAAGACCACGT
ATCG-3
0
; HAL-E414Q(–): 5
0
-CGATACGTGGTCTTG
CTGGTTGGC-3
0
; HAL-Q277A(1): 5
0
-CGACAAGGT
C
GCGGACCCGTACTCG-3
0
; HAL-Q277A(–): 5
0
-CGA
GTACGGGTCC

genes for wild-type HAL and HAL mutants were cultured
and HAL was purified as described previously [6].
SDS/PAGE and Western blot analysis
SDS/PAGE was carried out according to Laemmli [17]
using 10% polyacrylamide gels. The gels were stained with
Coomassie Brillant Blue R250. Western Blot analysis was
performed following a previously described method using
nitrocellulose blotting filters [18,19]. Wild-type HAL and
mutants were detected with rabbit polyclonal antibodies
raised against HAL from Pseudomonas putida (the antibody
was a generous gift from G. Mu
¨
nscher, Behringwerke AG,
Marburg, Germany).
Enzyme assay and protein determination
HAL activity was measured spectrophotometrically at 25 8C
following the formation of trans-urocanate at 277 nm. The
assay was performed in 1-cm quartz cuvettes by modifi-
cation of the method described in [20] with enzyme
concentrations varying between 1 and 25 mg for active
enzymes and between 0.1 and 1 mg for less active mutants.
The enzyme was preincubated at 25 8C for 5 min in 950 mL
0.1
M sodium pyrophosphate pH 9.3 supplemented with
10 m
M ZnCl
2
and 2 mM glutathione. Reaction was started
by adding 50 mL of a 0.5-
ML-histidine solution. Wild-type

L-cysteine was carried out in
1 cm quartz cuvettes as described earlier [10,11,27]. A total
Fig. 1. Mechanism for the formation of MIO by
cyclization of an intramolecular 142ASG144
tripeptide.
6012 D. Ro
¨
ther et al. (Eur. J. Biochem. 268) q FEBS 2001
of 0.75 mg (3.5 nmol) enzyme was dissolved in 1.0 mL
50 m
M NaHCO
3
/Na
2
CO
3
buffer pH 10.5. Inhibition was
started by addition of
L-cysteine to a final concentration of
10 m
M. Inactivation was controlled spectrophotometrically
in a Cary 3E spectrophotometer (Varian), following the
increase in absorbance at 338 nm during 50 min in intervals
of 10 min to get repetitive overlays of the absorption spectra.
For determining the time dependence of the inactivation
by activity measurements 12 nmol enzyme was dissolved in
2mL50m
M NaHCO
3
/Na

WINDOWS 95 or WINDOWS 98. For
molecular mechanics, a switched smoothing function which
gradually reduced nonbonding interactions to zero from
10 A
˚
inner radius to 14 A
˚
outer radius, was applied.
Otherwise, all calculations were performed by using default
settings of the program packages.
Analysis of the X-ray structure of the HAL homotetramer
(PDB code: 1B8F) showed that Ser143 is fully covered by
residues of three monomer subunits within a global area of
25 A
˚
radii. This part (representing 475 amino-acid residues,
a number which is comparable to the 509 amino-acid resi-
dues size of a monomeric HAL unit, together with structure
waters, a glycerol molecule and a sulfate anion) was cut off
from the full HAL homotetramer structure and used for
modelling the substrate free and substrate incorporating
states of the active site by
MM1 calculations of the
HYPERCHEM package [28]. All the mutated residues were
found within 12 A
˚
radii around the methylene carbon of
MIO formed from Ser143. Therefore the outside sphere
between 12 and 25 A
˚

ecules (hydrogen bonded to the imidazole N of H83 and to
the carbonyl O of Asn195) and the sulfate anion, all of
which are present in the experimental X-ray structure of
HAL in the close vicinity of the MIO methylidene moiety.
The atomic pairs used for this fit were: H83 coordinating
water O , imidazolyl-N1 of the histidine, Asn195
coordinating water O , NH
3
1
of histidine, and S atom
of the sulfate anion , carboxylate C of histidine. After
docking, the sulfate ion and the two water molecules were
deleted and the structure containing the zwitterionic
L-histidine substrate was optimized using the MM1 method
of the
HYPERCHEM [28] program (Fig. 2A).
The cationic intermediate state was obtained by con-
structing a single bond between the
L-histidine imidazole C5
and MIO methylidene C atoms, correcting the atom and
bond types and orders, and relaxing the structure by
MM1
optimization (Fig. 2B).
The trans-urocanate/ammonia binding model was
obtained from the cationic intermediate model by breaking
the appropriate bonds, correcting the atom and bond types and
orders, and optimizing the structure by the
MM1 method
(Fig. 2C).
Calculations of electronic spectra of different forms of a

Fig. 4 Active site of HAL.
6014 D. Ro
¨
ther et al. (Eur. J. Biochem. 268) q FEBS 2001
monomeric size. Western Blot analysis showed that all
enzyme variants were detected by the anti-HAL Ig.
E. coli BL21 (DE3) used as host did not show any HAL
activity. A search in the Swiss-Prot data bank for sequences
homologous to HAL from various sources was negative.
Purification of wild-type HAL and HAL mutants resulted
in yields varying from 5 to 80 mg pure enzyme per L cell
culture. After purification the turnover number or k
cat
of
recombinant wild type HAL was 86 s
21
which is in
agreement with a specific activity of 24 U
:
mg
21
previously
reported [6].
Characterization of the mutants by kinetic measurements
Steady state kinetic parameters of HAL mutants were
measured at substrate concentrations varying from 0.5 to
35 m
ML-histidine. Comparison of the K
m
values revealed

single mutant C273A divided by k
cat
of HAL double
mutants) are listed. The factors show to what extent the
double mutants are less active in relation to the single
mutant C273A. HAL mutant C273A/R283K shows < 20
times lower activity compared to HAL mutant C273A,
whereas a substitution of this arginine by isoleucine leads to
a larger decrease of activity (1640 times less active than
mutant C273A). This indicates that a noncationic residue at
that position results in a more severe decrease of activity.
Substitution of Y53, which is positioned in the neighbour-
hood of R283 at the active site, leads to more dramatic
effects. Exchange to phenylalanine results in a 2650-fold
less activity compared to the single mutant C273A. These
data indicate that this region in the active site may be
responsible for coordination of a cationic group of
L-histidine that is located near an anionic group of the
substrate. We propose therefore that the neighbouring resi-
dues R283 and Y53 coordinate the carboxylic and amino
group of the substrate
L-histidine, respectively. Based on the
X-ray structure of HAL (Fig. 4) in Fig. 2, models for
binding of
L-histidine (Fig. 2A), the cationic intermediate
formed by attack of C5 of the imidazole moiety of
L-histidine at the methylidene carbon of MIO (Fig. 2B), and
trans-urocanate and ammonia (Fig. 2C) at the active site of
HAL are shown which explain possible functions of some
active site residues. Residues Q277 and F329 (see Fig. 4)

cat
values (s
21
) were determined with the molecular mass M
r
¼ 53 593 for one subunit of the tetrameric HAL. Determination of protein
concentration was carried out according to Warburg and Christian [22,23], Murphy and Kies [24], Groves et al. [25] and Smith et al. [26].
K
m
(mM) k
cat
(s
21
) k
catC273A
/k
catmut
ratio
Wild-type HAL 3.9 ^ 0.9 86 ^ 6
C273A HAL 18 ^ 318^ 11
C273A/R283I HAL 18 ^ 4 0.011 ^ 0.001 1640
C273A/R283K HAL 4.1 ^ 0.7 0.79 ^ 0.03 20
C273A/Y53F HAL 8 ^ 1 0.0068 ^ 0.0004 2650
C273A/E414A HAL 6.1 ^ 0.7 0.00086 ^ 0.00007 20 930
C273A/E414Q HAL 1.7 ^ 0.9 0.053 ^ 0.0025 339
C273A/Y280F HAL 8 ^ 1 0.32 ^ 0.01 55
C273A/N195A HAL 3 ^ 1 0.018 ^ 0.001 1000
C273A/Q277A HAL 7 ^ 2 0.14 ^ 0.01 125
C273A/F329A HAL 4.4 ^ 0.7 0.18 ^ 0.01 100
C273A/H83L HAL 1.2 ^ 0.4 0.001 ^ 0.0002 18 000

cationic intermediate suggests that the imidazolyl N1-H
of the substrate may polarize the MIO group by partial
protonation of its carbonyl oxygen (Fig. 2B). This partial
protonation facilitates the electrophilic attack at the methyl-
ene moiety of MIO by decreasing of the electron density in
the p system of the C¼C double bond (Fig. 5).
Calculation of the UV spectra of MIO and the energy of
putative intermediate states
For estimating the degree of polarization of the MIO moiety
in the substrate free state of HAL by partial protonation of
Fig. 6. Arrangement of H83 and substrate imidazole in the cationic
intermediate model of HAL compared to experimental Zn
21
com-
plex found in human carbonic anhydrase II (PDB code: 1CRA) [31].
Table 2. Distances in models of the HAL active site. Selected
distances (measured in A
˚
) in models for the zwitterionic
L-histidine
binding (a), for the cationic intermediate containing (b), and for the
trans-urocanate/ammonia binding (c) state of HAL’s active site are
listed.
Atomic pairs Model a Model b Model c
S142
C3
–His
C4
0
4.43 1.53 3.89

–His
O1
2.84 2.87 4.55
Q277
N4
–His
O1
4.31 4.04 6.32
R283
NH1
–His
O
0
1
4.39 4.04 4.79
R283
NH2
–His
O
0
1
3.68 3.16 3.00
Fig. 7. Mechanism for the formation of the
338 nm chromophore by irreversible
inactivation of HAL with
L-cysteine.
6016 D. Ro
¨
ther et al. (Eur. J. Biochem. 268) q FEBS 2001
polar amino-acid residues, the electronic spectrum of this

and with the assistance of Y280 may provide the enzymic
base designed to abstract the activated b proton of the
substrate. Y53, R283 and Q277 might be involved in
anchoring the carboxylate moiety of the substrate or the
cationic intermediate (Fig. 2A,B). Inspection of the whole
HAL tetramer reveals that residue Y53, which showed the
most dramatic effect on the reaction rate among these three
residues, is located at the edge of a channel through which
the substrate can enter into or the product can be released
from the active site.
The calculated total energies for the zwitterionic
L-histidine binding (Fig. 2A) and cationic intermediate
binding models (Fig. 2A) were similar, whereas
< 46 kJ
:
mol
21
lower total energy was obtained for
Fig. 8. Inactivation of HAL with L-cysteine to a final concentration
of 10 m
M, and an enzyme concentration of 0.75 mg (3.5 nmol) in
1 mL. For further details see Experimental procedures. Inactivation of
HAL mutant C273A and repetitive scans between 0 and 50 min after
supplementation of
L-cysteine (A). Inactivation of HAL mutant C273A/
Y280F and repetitive scans between 0 and 50 min (B). Inactivation of
HAL mutant C273A/Y280F and C273A/H83L 20 and 48 h after
supplementation of
L-cysteine, respectively (C).
Fig. 9. Inactivation with L-cysteine and enzyme assay between 0

tration of 10 m
M in slightly basic solution and in the
presence of O
2
. Under these conditions, both wild-type HAL
and HAL mutant C273A show an increase in absorbance at
338 nm during 50 min as previously described [11]. The
chromophore is generated by nucleophilic attack of the
thiolate anion of cysteine at the MIO group followed by
oxidation and intramolecular S-to-N rearrangement as
recently proposed (Fig. 6) [32,33]. In Fig. 8A repetitive
scans of the single mutant C273A following the inactivation
with
L-cysteine are shown. During 50 min, an absorbance
maximum develops that is located around 338 nm. Some
double mutants showed different behaviour upon treatment
with
L-cysteine. The mutant C273A/F329A did not show an
absorbance maximum upon treatment with
L-cysteine even
after 24 h of incubation. In the case of the HAL mutant
C273A/Y280F and mutant C273A/H83L there was a slower
increase in absorbance but after 20 and 48 h, respectively, a
chromophore around 338 nm appeared also in these cases
(Fig. 8C). These results indicate the presence of a MIO at
their active sites, but in a less reactive form. Concomittant
with the formation of a new chromophore the activity of the
enzyme decreases irreversibly. After addition of
L-cysteine,
the activity of the enzymes dropped very quickly and in

enzymatic activity by removal of such ions and the slight
activation at their presence might be explained by assuming
their interaction with H83 and the substrate histidine
(Fig. 6). In contrast no metal-ion effect has ever been
observed on the PAL reaction. This is in agreement with
the lack of histidine in a similar position in all PAL
sequences.
ACKNOWLEDGEMENTS
We thank Prof. G. E. Schulz and Dr T. F. Schwede (University of
Freiburg, Germany) for the cooperation in the work on HAL and PAL
and the production of two HAL mutants. The work was supported by the
Deutsche Forschungsgemeinschaft and the Fonds der Chemischen
Industrie. D. R. thanks the Land Baden-Wu
¨
rttemberg for a scholarship
for graduate students. L. P. thanks the Hungarian OTKA (T-033112) for
financial support. We thank A. Sigrist for help with the figures and
S. Vollmer for technical assistence.
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