Insights into the reaction mechanism of
Escherichia coli
agmatinase
by site-directed mutagenesis and molecular modelling
A critical role for aspartate 153
Mo
´
nica Salas
1
, Rolando Rodrı
´
guez
2
, Nelia Lo
´
pez
2
, Elena Uribe
1
, Vasthi Lo
´
pez
1
and Nelson Carvajal
1
1
Departamento de Biologı
´
a Molecular, Facultad de Ciencias Biolo
´
gicas, Universidad de Concepcio

and orientation of a metal-bound hydroxide for optimal
attack on the guanidinium carbon of agmatine. Thus, the
disruption of this hydrogen bond is the likely cause of the
greately decreased catalytic efficiency of the D153N variant.
Keywords: agmatinase; Asp153; site-directed mutagenesis;
homology-modelling; E. coli.
Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) cata-
lyses the hydrolysis of agmatine to putrescine and urea [1].
Agmatine, which results from decarboxylation of arginine
by arginine decarboxylase [2], is a metabolic intermediate in
the biosynthesis of putrescine and higher polyamines [1] and
may have important regulatory roles in mammals [3–5].
Agmatinases from Escherichia coli and human tissues,
and putative agmatinases from Synechocystic sp. Schizo-
saccharomyces pombe and Bacillus subtilis, have been cloned
and the deduced amino acid sequences indicate their
homology to all sequenced arginases [4–7]; all these enzymes
catalyse an hydrolytic reaction with production of urea. The
question arises therefore as to whether a similar or identical
mechanism is involved in catalysis by these enzymes, which
apparently evolved from a single primordial protein [6,7]. In
this context, both enzymes exhibit an absolute requirement
for Mn
2+
for catalytic activity [8,9]; the well established
requirement of a binuclear metal cluster for full catalytic
activity of arginase [8] is probably also valid for agmatinase
[9]. This is reinforced by the fact that residues known to be
metal ligands in arginase are strictly conserved in the
sequence of agmatinase [7]. Moreover, a critical role for one

enzymes and reagents for PCR were obtained from
Promega. The plasmid pKA5, bearing the speB gene of
E. coli agmatinase, was kindly supplied by S. Boyle (Vir-
ginina Polytechnic Institute and State University). The
pQE60 E. coli expression vector and the Ni-nitrilotriacetic
acid resin were obtained from Qiagen, and synthetic
Correspondence to N. Carvajal, Departamento de Biologı
´
a Molecular,
Facultad de Ciencias Biolo
´
gicas, Universidad de Concepcio
´
n,
Casilla 160-C, Concepcio
´
n, Chile. Fax: + 56 41 239687;
E-mail:
(Received 5 June 2002, revised 9 September 2002,
accepted 12 September 2002)
Eur. J. Biochem. 269, 5522–5526 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03255.x
nucleotide primers from the Centro de Estudios Avanzados
(Universidad de Chile).
Enzyme preparations and enzyme assays
Bacteria were grown with shaking at 37 °C in Luria broth in
the presence of ampicillin (100 lgÆmL
)1
). The wild-type and
D153N agmatinase cDNAs were directionally cloned into
the histidine tagged pQE60 E. coli expression vector and the

and a 3¢ complementary primer corresponding to nucleo-
tides 448–468 of agmatinase with a CfiT substitution at
nucleotide 457 (sequence: 5¢-CGCATAGGTAT
TG
GTGTGGGC-3¢). Similarly, the second PCR product was
obtained using the 5¢ sense primer corresponding to
nucleotides 448–468 of agmatinase with a GfiA substitu-
tion at nucleotide 457 (sequence: 5¢-GCCCACACC
AAT
ACCTATGCG-3¢) and the 3¢ complementary primer 5¢-AT
TAATGGCATGCTTTACCCGT-3¢.UsingthePCR
products of agmatinase with the GfiAandCfiT substi-
tutions in the coding and noncoding strands, respectively,
and using the 5¢ and 3¢ primers mentioned above, the full
length agmatinase cDNA coding for the D153N mutant
was generated by a second round of PCR. The expected
mutation was confirmed by DNA sequence analysis. That
no unwanted mutations had been introduced during the
mutagenesis process was verified by automated sequencing.
The H163F E. coli agmatinase was obtained as described
previously [10].
Fluorescence spectra
Fluorescence measurements were made at 25 °Cona
Shimadzu RF-5301 spectrofluorimeter. The protein concen-
tration was 30 lgÆmL
)1
and emission spectra were measured
with the excitation wavelength at 295 nm. The slit width for
both excitation and emission was 1.5 nm, and spectra were
corrected by subtracting the spectrum of the buffer solution

derived from the structural superposition of two arginase
structures (rat liver and B. caldovelox arginases, PDB id
1RLA and 1CEV, respectively). The agmatinase
SPEB_ECOLI sequence, obtained from the Swissprot
database (accession number P16936), was separately aligned
with the sequences of 1CEV and 1RLA and pasted into the
structural alignment. The alignment was then corrected by
hand. Since the sequences of the two arginases and
agmatinase greately differ in the N terminal region, the
agmatinase and rat liver arginases were stripped of the first
32 and five amino acids, respectively. The location of the
gaps was optimized by repetitive modelling, shuffling those
aminoacids that were not conserved in the structural
alignment, to obtain the shortest possible Ca-Ca distance.
The resulting sequence alignment, along with the secondary
structure elements, is shown in Fig. 1. All loops that were
different due to insertions or deletions in the agmatinase
sequence were modelled using the DGLOOP set of options
in
WHATIF
; the whole loops and the two connecting amino
acids at the beginning and the end, were mutated to glycines
and, after remodelling back to their side chains in agma-
tinase, the whole hydrogen bond network was optimized.
The position of the active site manganese ions was calculated
using the averaged distance template of 1CEV and 1RLA,
and then a full hydrogen bond network optimization was
performed again. Agmatine was added to agmatinase using
the same superposition matrix calculated for manganese
ions, in order to conserve the ligand-manganese distances.

for agmatine (1.6 ± 0.1 m
M
) and the K
i
for competitive
inhibition by putrescine (12 ± 2 m
M
), were also essentially
equal for wild-type and D153N agmatinases. It is clear
therefore that altered interactions with the substrate or a
significantly altered affinity for the activating metal ion, are
not the explanations for the greatly decreased catalytic
activity of the D153N variant.
To evaluate possible structural changes that may result
from mutation, wild-type and D153N enzymes were com-
pared by using fluorescence and CD spectrometry. The
tryptophan emission fluorescence spectra were not altered
by mutation (k
max
¼ 342 nm), indicating that the environ-
ment of tryptophan residues is essentially conserved in the
mutant enzyme. On the other hand, the absence of major
differences in the CD spectra of the wild-type and D153N
agmatinases indicates that mutation had no effect on their
respective secondary structures (Fig. 3). As an example, the
percentage values calculated for the a-helix were 22.3 and
21.5% for wild-type and D153N enzymes, respectively.
Therefore, based on the criteria used in this study, we may
discard a gross structural change as the explanation for the
lower catalytic activity of D153N agmatinase.

andassayedintheabsenceofaddedmetal
ion.
Fig. 1. Structural sequence alignment of
B. caldovelox arginase (1cev), E. coli agma-
tinase (AUH) and rat liver arginase (1rla). H, S,
T and 3 stands for a-helix, strand, turn and
3
10
-helix. Conserved residues are marked by
an asterisk.
5524 M. Salas et al. (Eur. J. Biochem. 269) Ó FEBS 2002
differed in these areas. A more specific difference concerned
a loop located at the entrance of the active site cleft, and
defined by residues 124–141 in the sequence of the bacterial
arginase. Whereas the backbone and some of the side chains
are very precisely conserved in both arginases, the agma-
tinase loop was shorter when compared with the same
region of the arginases (Fig. 4). Since the arginase loop
contains residues that interacts with the a-carboxylate
group of the substrate arginine [13], and this is the part of
the molecule that makes arginine different from agmatine, it
seems reasonable to assume that differences in this loop area
are key factors in determining the difference in substrate
specificity between arginase and agmatinase. This aspect is
presently under investigation in our laboratory. In any case,
despite the structural differences between agmatinase and
arginase, agmatine and arginine were fixed in essentially the
same position in the corresponding active site. The same
position for the scissile guanidine carbon of the substrates,
with respect to the metal ions and conserved, catalytically

unaltered interaction of the D153N variant with the metallic
cofactor.
In the modelled structure, the noncoordinated carboxy-
late oxygen atom of Asp153 is within hydrogen-bonding
distance of the metal-bound water molecule. This is
interesting if one considers a catalytic mechanism involving
a nucleophilic attack of a metal-bound hydroxide on the
guanidinium carbon of agmatine [8,9]. By donating an
hydrogen bond to Asp153, the nucleophile would be
stabilized and oriented for optimal catalysis. On this basis,
the disruption of this stabilizing hydrogen bond, due to the
replacement of the carboxylic oxygen by the amide nitrogen,
would be expected to result in a less efficient catalysis by the
metal-bound hydroxide in the D153N mutant enzyme. The
low, but significant catalytic activity of the mutant indicates
that, even in the absence of the hydrogen bond to the
noncoordinating carboxyl oxygen, the metal-bound
hydroxide could still serve as a catalytic nucleophile,
although considerably less efficiently. Further studies are,
evidently, required to clarify this aspect. The proposed role
for the critical Asp153 in E. coli agmatinase reinforces the
relationships between this enzyme and the evolutionary
related arginase. It would be, thus, of interest to examine
the effects of the corresponding aspartate to asparagine
mutation in this enzyme.
ACKNOWLEDGEMENTS
This research was supported by Grant 2990049 from FONDECYT and
P.I. 98.031.076-1.0 from the Direccio
´
ndeInvestigacio

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(1996) Structure of a unique binuclear manganese cluster in argi-
nase. Nature 382, 554–557.
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´
pez,V.,Salas,M.,Uribe,E.,Herrera,P.&Cerpa,
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´
pez, V., Herrera, P.
& Cerpa, J. (1999) Chemical modification and site-directed
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