Contribution of Lys276 to the conformational flexibility of the active
site of glutamate decarboxylase from
Escherichia coli
Angela Tramonti
1
, Robert A. John
2
, Francesco Bossa
1
and Daniela De Biase
1
1
Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’ and Centro di Studio sulla Biologia Molecolare del CNR, Rome, Italy;
2
Cardiff School of Biosciences, Cardiff, UK
Glutamate decarboxylase is a pyridoxal 5¢-phosphate-
dependent enzyme responsible for the irreversible a-decar-
boxylation of glutamate to yield 4-aminobutyrate. In
Escherichia coli, as well as in other pathogenic and non-
pathogenic enteric bacteria, this enzyme is a structural
component of the glutamate-based acid resistance system
responsible for cell survival in extremely acidic conditions
(pH < 2.5). The contribution of the active-site lysine
residue (Lys276) to the catalytic mechanism of E. coli
glutamate decarboxylase has been determined. Mutation of
Lys276 into alanine or histidine causes alterations in the
conformational properties of the protein, which becomes less
flexible and more stable. The purified mutants contain very
little (K276A) or no (K276H) cofactor at all. However,
apoenzyme preparations can be reconstituted with a full
complement of coenzyme, which binds tightly but slowly.

It also facilitates the proton transfers essential to many
B6-dependent reactions [3–10]. In the amino acid decar-
boxylases so far investigated, the corresponding lysine
appears not to be involved in reprotonation after
decarboxylation, but mainly to play a role in the initial
transaldimination, in proper positioning of the a-carb-
oxylate for decarboxylation and in product release
[11,12].
Bacterial glutamate decarboxylase (Gad, E.C. 4.1.1.15) is
one of the structural components of the glutamate-based
acid resistance system, responsible for acid survival of
enteric pathogens, such as Escherichia coli, Shigella flexneri
and Listeria monocytogenes [13–15], and of other nonpatho-
genic bacteria [16,17]. E. coli synthesizes two Gad isoforms,
GadA and GadB, 98% identical in amino acid sequence
and biochemically indistinguishable [18,19]. Gad catalyses
the irreversible a-decarboxylation of
L
-glutamate to yield 4-
aminobutyrate and CO
2
. It has been suggested that in this
enzyme the active-site lysine is involved in the protonation
of the quinonoid intermediate at C-4¢ during the abortive
decarboxylation–transamination reaction, while a histidine
has been proposed as the residue responsible for the
protonation at C-a which occurs during the physiological
decarboxylation reaction [20]. Site-directed mutagenesis
established that His167 and His275, likely candidates as
proton donors, are not responsible for the reprotonation

nidineÆHCl, 2,2,2-trifluoroethylamine, aminoacetonitrile
bisulfate and Gabase were from Sigma. Other chemicals
were from Merck.
Site-directed mutagenesis
Site-directed mutagenesis was performed by overlap exten-
sion polymerase chain reactions [22], following the proce-
dure described in Tramonti et al.[21].Mutagenicprimers
were 5¢-GGCCATGCATTCGGTCTG-3¢,fortheGadB-
K276A mutant, 5¢-GGCCATCACTTCGGTCTG-3¢,for
the GadB-K276H mutant, and their complementary
sequences. Fragments EcoRV/HindIII, generated by digest-
ing the amplicons from the second polymerase chain
reaction, were subcloned into pQgadB [19]. The newly
inserted fragments of plasmid pQgadBK276A and
pQgadBK276H were sequenced on both strands.
Purification of mutant forms of Gad
Expression and purification of mutant enzymes were as
described for wild-type enzyme [19]. The E. coli strain
JM109(pREP4), known to produce low levels of endo-
genous GadA/B was used as host [19]. Preparations of the
mutant enzymes were treated with NaBH
3
CN to inactivate
any wild-type enzyme present.
Calorimetric and spectroscopic analyses
Thermal unfolding of GadB-K276A and GadB-K276H
(1.5–2.0 mgÆmL
)1
) was analyzed under nitrogen pressure
on a MicroCal MC-2D differential scanning calorimeter

À Abs
E
Abs À Abs
E
À 1 ¼
10
ÀnpK
10
ÀnpH
ð1Þ
where Abs
HnE
and Abs
E
are the absorbances of completely
protonated and unprotonated forms of enzyme, K is the
intrinsic dissociation constant and n is the number of
protons involved in titration.
The change in absorbance observed in the reaction of
GadB-K276A and GadB-K276H mutants with glutamate
was analyzed with Eqn (2) which describes two consecutive
irreversible reactions of the type A !
k
1
B !
k
2
C.
b ¼ a
0

RESULTS
Physical properties of mutant enzymes
Yields of the mutant enzymes, GadB-K276A and GadB-
K276H, after the standard purification, i.e. in absence of
added PLP, were 50 mgÆL
)1
of bacterial culture, as for
wild-type GadB [19]. The mutant forms were stable for
several months at 4 °C. As judged by CD spectroscopy in
the far-UV region, mutations did not affect the overall
protein conformation. The transition temperature of
reconstituted GadB-K276A was 62.3 °C, suggesting that
this mutant enzyme adopts a significantly more stable
conformation than wild-type GadB (51 °C). The transi-
tion temperature of GadB-K276H (55.3 °C) was only
slightly higher than that of the wild-type enzyme (Fig. 1).
In support of the above observation, limited proteolysis
by trypsin showed that GadB-K276A is more resistant to
proteolytic degradation than the wild-type enzyme (data
not shown).
Spectral properties of GadB-K276A and -K276H
mutants
Absorption spectra of purified GadB-K276A showed
maxima at 280 and 328 nm (Fig. 2A). However, the specific
absorbance at 328 nm due to the cofactor was significantly
lower than that of the wild-type enzyme and, correspond-
ingly, the amount of PLP released by NaOH treatment was
only 10% of that expected for a fully saturated holoenzyme.
Nevertheless, the small amount of cofactor present was not
displaced by either gel filtration in 0.5

(750 l
M
). Unbound cofactor was removed by extensive
dialysis against 0.1
M
sodium acetate, pH 4.6. The recon-
stituted mutant enzyme (Fig. 2A) contained one molecule
of PLP per monomer, as judged by NaOH treatment. The
absorption spectrum above 320 nm fitted well to the sum of
two log normal curves having k
max
values of 330 nm and
388 nm (Fig. 2B). The great majority of the coenzyme was
present as 330 nm-absorbing chromophore, the corres-
ponding peak being broader, but less intense, in the GadB-
K276H mutant enzyme (Fig. 2B). Continuous monitoring
of the absorbance changes associated with reconstitution of
GadB-K276A indicated that the absorbance decrease at
388 nm (free PLP) and the increase at 330 nm were biphasic
(data not shown) and the curve fitted well to the sum of
two exponentials (k
1
¼ 0.58 ± 0.001 min
)1
; k
2
¼ 0.059 ±
0.002 min
)1
) with the more rapid phase accounting for 70%

absorption spectra of GadB-K276A and GadB-K276H mutants. The
solid lines are those of best fit to the sum of two log normal curves [32]
having k
max
values of 330 nm and 388 nm. Only one in three of the
data points collect for the GadB-K276A (d) and GadB-K276H (j)
absorption spectra is shown. (C) CD spectra of wild-type and mutant
enzymes. The CD spectra of wild-type GadB (solid line), and of GadB-
K276A (dotted line) and GadB-K276H (dashed line) mutants, each at
a concentration of 184 l
M
, were determined in 50 m
M
sodium acetate,
pH 4.6, containing 0.1 m
M
dithiothreitol.
Fig. 1. Differential scanning calorimetry of wild-type GadB and active-
site lysine mutants. Thermal denaturation profiles of GadB wild-type
(solid line), of GadB-K276A mutant (dashed line) and GadB-K276H
mutant (dotted line). Protein samples (1.5–2.0 mgÆmL
)1
)werein0.1
M
sodium acetate, pH 3.6, containing 0.1 m
M
dithiothreitol.
Ó FEBS 2002 Role of Lys276 in E. coli glutamate decarboxylase (Eur. J. Biochem. 269) 4915
change and it is clear from the pH profile that multiple
protons are involved in the transition (Fig. 3A, inset) [26]. In

fluorescence of the protein. It is likely that the second, at
380 nm, is due to energy transfer to the 330-nm absorbing
form of PLP at the active site. At 2
M
guanidineÆHCl the
emission spectrum of both mutants showed exclusively a
peak at 332 nm, because the PLP in the active site has been
converted into a form mainly absorbing at 388 nm. In the
range 2–5
M
guanidineÆHCl the change in fluorescence
emission spectra indicated that the unfolding profiles of
wild-type and mutant enzymes are superimposable, with the
transition point (50% unfolding) centered at 3.4
M
guani-
dineÆHCl. Upon unfolding, a blue-shifted emission maxi-
mum at 360 nm in both wild-type and mutant enzymes was
observed (Fig. 4).
Reaction with glutamate
Addition of 20 m
M
glutamate to GadB-K276A produced
an increase in absorbance at 412 nm and a decrease at
328 nm each with the same half-time of approximately 2 h
(Fig. 5A). The change was characterized by an isosbestic
point at 342 nm. The 412 nm contribution was completely
abolished by adding NaCNBH
3
, a reagent known to reduce

HnE
¼ 0.1058 ±
0.0007 and n ¼ 5.1 ± 0.4. (B) Absorption spectra of GadB-K276H
(10.3 l
M
) were determined as above. In the inset, the pH variation at
388nmisrepresented.ThesolidlineisthatofbestfittoEqn(1)
(Materials and methods), with pK ¼ 5.60 ± 0.01, Abs
E
¼ 0.032 ±
0.001, Abs
HnE
¼ 0.0060 ± 0.0006 and n ¼ 8.4 ± 4.6. (C) Absorption
spectra of GadB-K276H mutant (19 l
M
) measured in the presence of
0, 0.1, 0.2, 0.4, 0.6, 1 and 2
M
guanidineÆHCl in 0.1
M
sodium acetate,
pH 4.6. The pH- and guanidine-dependent absorbance changes in the
GadB-K276A mutant were identical with those in GadB-K276H
mutant, and therefore the data are omitted.
4916 A. Tramonti et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Moreover, no pH increase was detected during the reaction
with glutamate when using the pH indicator bromocresol
green in an unbuffered solution, thus indicating that there
was no consumption of protons. Treatment of the reaction
mixture with 0.2

¼ 5.3) [7] were added to the enzyme in
presence of sodium glutamate.
When 1
M
2,2,2-trifluoroethylamine or 0.2
M
amino-
acetonitrile bisulfate were included in the reaction mixture
containing 20 l
M
GadB-K276A and 20 m
M
sodium
glutamate, the enzyme underwent spectral changes identical
to those already described, but the increase in absorbance at
412 nm occurred approximately six times faster. The devel-
opment of turbidity however, prevented analysis of the later
phases of the reaction. Even in the presence of exogenous
amines 4-aminobutyrate production was undetectable (data
not shown).
When both mutant enzymes were incubated with
glutamate in the presence of a low concentration of
guanidineÆHCl (0.4
M
), spectral changes identical to those
previously described occurred, even though the initial
spectrum was different, and at the end of reaction a
species absorbing at 340 nm could be detected (data not
shown).
DISCUSSION

guanidineÆHCl.
Ó FEBS 2002 Role of Lys276 in E. coli glutamate decarboxylase (Eur. J. Biochem. 269) 4917
of the holoenzyme by 11 °C, makes it resistant to tryptic
hydrolysis and prevents the precipitation observed with the
wild-type apoenzyme demonstrates that the absence of this
lysine residue, either as an aldimine with the cofactor or as a
protonated primary amine in the apoprotein, makes the
protein less flexible. The same reduced flexibility seems
likely to account for the slow, but ultimately tight, binding
of PLP to the apoenzyme in vitro and for the fact that
preparations of the mutant enzymes are always largely as
apoenzyme. Similarly, inflexibility of the protein would also
explain the observation that PLP, bound to the mutant
enzymes, forms an aldimine with glutamate much more
slowly than free PLP.
The observation that the mutant apoenzymes are able to
form stable holoenzymes despite the absence of the active
site lysine residue shows that, as in other decarboxylases
[11,12], non–covalent interactions between protein and
cofactor are sufficient to ensure tight binding. This is also
in line with the finding that His275 contributes to cofactor
binding via an ionic interaction with the phosphate group of
PLP [21].
Because the mutant forms of GadB cannot form an
internal aldimine, it is not surprising that the 420 nm
chromophore, characteristic of the wild-type enzyme at
pH 4.6, is absent. However, the spectrum of PLP bound to
the Lys276 mutants is quite different from the spectrum of
the same compound when it is free in solution. Absorption
bands at 388 nm and 330 nm are present in the spectra of

to the ionization of a single proton [30]. It has been
suggested that in wild-type Gad, the ionization responsible
for the absorbance change does not take place on the
internal aldimine and much evidence indicates that the
change in spectrum of the wild-type enzyme is due to a
conformational transition in the protein induced by shifting
the pH [31]. It seems very likely that the pH-dependent
changes that occur in the spectrum of the mutants under
investigation in the present work are due to the same pH-
induced conformational transition observed in the wild-type
and that the different forms of the cofactor present in wild-
type and mutant enzymes are recording the same event at
the active site. The observation that the pH-dependent
spectral changes occur in enzyme forms without the internal
aldimine demonstrates that the protonation responsible for
the absorbance changes and for activation of the wild-type
enzyme is not of the internal aldimine itself.
To explain the pH-dependent occurrence of the
330 nm-absorbing chromophore, it has been proposed
that, in the wild-type enzyme, high pH induces the
formation of an aldamine between the internal aldimine
and an enzyme cysteine residue and that the low pH
conformation favors the unsubstituted internal aldimine
[31]. However, because in the mutant enzymes the high
pH favors the 388 nm-absorbing unsubstituted aldehyde,
formation of a covalent bond between PLP and a cysteine
side-chain can be excluded as the basis of the pH-
dependent absorbance changes observed with the Lys276
mutant enzymes. An explanation that unites observations
from both wild-type and mutant enzymes is that the pH-

-glutamate and forming an
external aldimine. The increase in absorbance at 412 nm
observed when GadB K276A was mixed with glutamate,
together with the observation that this chromophore
converted to one absorbing at 340 nm when NaCNBH
3
was added provides strong evidence that an external
aldimine is formed between the cofactor and the amino
acid. The linear dependence on glutamate concentration of
the observed rate constant governing this phase shows
that there is no detectable saturation of the mutant
enzyme with substrate, even at high concentrations. The
second order constant calculated from this experiment
(3.6 · 10
)3
±0.3 · 10
)3
M
)1
Æs
)1
) is much lower than
that calculated for the reaction between free PLP and
glutamate in 0.1
M
sodium acetate, pH 4.6 (1.47 ±
0.13
M
)1
Æs

dell’Istruzione, dell’Universita
`
e della Ricerca and from the Istituto
Pasteur-Fondazione Cenci Bolognetti (to DDB). The Centro di
Eccellenza di Biologia e Medicina Molecolare (BEMM), Universita
`
di Roma La Sapienza, is also acknowledged.
We thank Professor A. Giartosio for DSC measurements and
Professor D. Barra for critical reading of the manuscript.
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