Tài liệu Báo cáo Y học: Insights into the reaction mechanism of Escherichia coli agmatinase by

Insights into the reaction mechanism of Escherichia coli agmatinase

by site-directed mutagenesis and molecular modelling

A critical role for aspartate 153

Mo´ nica Salas1

, Rolando Rodrı´guez2

, Nelia Lo´ pez2

, Elena Uribe1

, Vasthi Lo´ pez1 and Nelson Carvajal1

1

Departamento de Biologı´a Molecular, Facultad de Ciencias Biolo´gicas, Universidad de Concepcio´n, Casilla 160-C, Concepcio´n,

Chile; 2

Center for Genetic Engineering and Biotechnology, Habana, Cuba

Upon mutation of Asp153 by asparagine, the catalytic

activity of agmatinase (agmatine ureohydrolase, EC

3.5.3.11)from Escherichia coli was reduced to about 5% of

wild-type activity. Tryptophan emission fluorescence (kmax

¼ 340 nm), and CD spectra were nearly identical for wild￾type and D153N agmatinases. The Km value for agmatine

(1.6 ± 0.1 mM) , as well as the Ki for putrescine inhibition

(12 ± 2 mM)and the interaction of the enzyme with the

required metal ion, were also not altered by mutation. Three￾dimensional models, generated by homology modelling

techniques, indicated that the side chains of Asp153 and

Asn153 can perfectly fit in essentially the same position in the

active site of E. coli agmatinase. Asp153 is suggested to be

involved, by hydrogen bond formation, in the stabilization

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 Mn2+ 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

conserved histidine residue (His163 in the sequence of

E. coli agmatinase)has been shown by chemical modifica￾tion and site-directed mutagenesis of human and rat liver

arginases [10,11] and E. coli agmatinase [12]; similar infor￾mation was deduced from X-ray crystallographic data for

arginase from Bacillus caldovelox [13].

Based on the crystal structure of rat liver arginase, it was

suggested that arginine hydrolysis involves the participation

of a metal-bound hydroxide, which is stabilized for optimal

nucleophilic attack at the substrate, by donating an

hydrogen bond to Asp128 [8,14,15]. In this connection,

the D128G variant of human liver arginase was described as

inactive [16,17], although the possible influence of structural

changes accompanying the mutation were not examined.

Since this aspartate is conserved among all sequenced

arginases and agmatinases [4–7], a critical role for the

equivalent residue in agmatinase (Asp153), may be reason￾ably expected. This expectation is supported by our present

findings of a markedly decreased activity of a D153N

variant of E. coli agmatinase. From the enzymic properties

of D153N agmatinase and a modelled structure, we

conclude that the lower activity of the mutant may be

ascribed to the loss of an acceptor hydrogen bond to a

metal-bound hydroxide, as a consequence of replacement of

a carboxylate oxygen with an amide group.

MATERIALS AND METHODS

Materials

All reagents were of the highest quality commercially

available (most from Sigma Chemical Co.)and were used

without further purification. Restriction enzymes, as well as

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: [email protected]

(Received 5 June 2002, revised 9 September 2002,

accepted 12 September 2002)

Eur. J. Biochem. 269, 5522–5526 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03255.x