Tài liệu Báo cáo Y học: Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling ppt

Insights into the reaction mechanism of Escherichia coli agmatinaseby site-directed mutagenesis and molecular modelling A critical role for aspartate 153 Mo´nica Salas1, Rolando Rodrı´gu

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´pez1and Nelson Carvajal1

1 Departamento de Biologı´a Molecular, Facultad de Ciencias Biolo´gicas, Universidad de Concepcio´n, Casilla 160-C, Concepcio´n, Chile;2Center 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 Kmvalue for agmatine

(1.6 ± 0.1 mM) , as well as the Kifor 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 pombeand 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 coliagmatinase)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

M A T E R I A L S A N D M E T H O D S

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 coliagmatinase, 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: ncarvaja@udec.cl

(Received 5 June 2002, revised 9 September 2002,

accepted 12 September 2002)

nucleotide primers from the Centro de Estudios Avanzados

(Universidad de Chile)

Enzyme preparations and enzyme assays

Bacteria were grown with shaking at 37C 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

histidine-tagged enzyme was expressed in E coli strain

JM109, following induction with 1 mM isopropyl

thio-b-D-galactoside The histidine-tagged enzymes were purified

to homogeneity by metal chelate chromatography over

Ni-nitrilotriacetic acid resin, according to the instructions of

the manufacturer A single protein band was detected by

SDS/PAGE of purified enzymes

Enzyme activities were determined by measuring the

formation of urea from agmatine in 50 mMglycine/NaOH

(pH 9.0) Urea was determined by a colorimetric method

with a-isonitrosopropiophenone [18] and protein

concen-trations were estimated by the method of Bradford [19],

with bovine serum albumin as standard Kinetic data were

analyzed by double reciprocal plots, and the Kivalue for

putrescine inhibition was determined from a replot of slopes

vs inhibitor concentration All lines were computer-fitted to

the appropriate equations

Site-directed mutagenesis

The D153N mutant form of E coli agmatinase was

obtained by a two-step PCR [20], using the plasmid

pKA5 containing the speB gene of E coli agmatinase as a

template A first PCR product was obtained using the 5¢

sense primer 5¢-AGTCCATCCATGGGCACCTTAG-3¢

and a 3¢ complementary primer corresponding to

nucleo-tides 448–468 of agmatinase with a CfiT substitution at

nucleotide 457 (sequence: 5¢-CGCATAGGTATTG

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¢-GCCCACACCAAT

ACCTATGCG-3¢)and the 3¢ complementary primer 5¢-AT

TAATGGCATGCTTTACCCGT-3¢ Using the PCR

products of agmatinase with the GfiA and CfiT

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 25C on a

Shimadzu RF-5301 spectrofluorimeter The protein

concen-tration was 30 lgÆmL)1and 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

(5 m Tris/HCl, pH 7.5)in the absence of protein

Circular dichroism

CD experiments were performed on a Jasco J-810 spectro-polarimeter thermostated at 22C CD spectra of wild-type and D153N mutant enzymes (5.5 lM)were measured in the range 200–250 nm, with a bandwidth of 1 nm and a scan speed of 50 nmÆmin)1 The buffer solution contained 5 mM

Tris/HCl (pH 7.5)and 2 mMMn2+ The reported spectra represents the averages of five repeat scans Spectra were smoothed and analysed for protein secondary structures by using the software package provided with the instrument Molecular modelling

The agmatinase structural model was obtained by homology methods, using the structure of B caldovelox arginase (PDB id: 1CEV)as a template and the modelling packageWHAT IF

[21] An amino-acid sequence identity of 29% was calculated for E coli agmatinase and B caldovelox arginase The sequence alignment used in the modelling experiment was 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

inWHATIF; 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

R E S U L T S A N D D I S C U S S I O N

By site-directed mutagenesis, a D153N mutant form of

E coli agmatinase was obtained The mutant enzyme retained about 5% of wild-type activity and it was equally active when assayed in the presence or absence of Mn2+ However, as shown in Fig 2, it was half inactivated by dialysis against 10 mMEDTA in 5 mMTris/HCl (pH 7.5) for 4 h at 4C and full recovery of enzyme activity was produced by incubation of the EDTA-treated species with

2 mMMn2+for 20 min at 37C; as expected, the effect of the metal ion was again reversed by EDTA These results indicate the presence of tightly and weakly bound mangan-ese ions in fully active species of the mutant enzyme

Identical behaviour was previously described, and

con-firmed here (Fig 2), for wild-type agmatinase and this was

interpreted as supporting the presence of a binuclear metal

center in the active site of fully activated agmatinase [9] The

Kmfor agmatine (1.6 ± 0.1 mM)and the Kifor competitive

inhibition by putrescine (12 ± 2 mM), 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 (kmax¼ 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

Since an experimentally derived structure is not yet available for any agmatinase, to further evaluate the consequences of the Asp153fiAsn substitution, we used a modelled structure of the E coli enzyme, constructed by using homology-modelling techniques and the 3D structure

of the binuclear form of B caldovelox arginase as a template Principal attention was given to the modeling of the active site The modelled structure was very similar to the template, with respect to the number and arrangement

of structural elements (a-helix and b-sheets), and one of the major differences concerned the surface loops However, as shown in Fig 4, B caldovelox and rat liver arginases also

Fig 2 Effect of added Mn2+and EDTA on the catalytic activity of

wild-type and D153N agmatinase The enzymes were assayed before

(Control)and after dialysis against 10 m M EDTA in 5 m M Tris/HCl

(pH 7.5)for 4 h at 4 C Enzyme activities were measured with and

without a previous incubation with 2 m M Mn 2+ for 20 min at 37 C.

Activities are expressed as percentage of the corresponding control not

preincubated with Mn 2+ and assayed in the absence of added metal

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.

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 important residues, agree with a similar, if not identical, mechanism for both enzymes

Upon replacement of Asp153 with asparagine, the whole topology of the active site was found to be conserved in agmatinase In the modelled active site structure, the side chains of Asp153 and Asn153 can be accommodated at essentially the same position, with the whole distance network remaining almost intact (Fig 5) Moreover, one of the carboxylate oxygens of Asp153 and the carboxamide oxygen of Asn153 are positioned in such a way as to allow metal coordination interaction with one of the manganese ions This, together with the fact that the positions of other

Fig 4 A superimposition of the structures of B caldovelox arginase

(red), rat liver arginase (yellow) and the modelled structure of E coli

agmatinase (blue) Note the shorter extension, in the case of E coli

agmatinase, of the loop indicated by the letter a.

Fig 5 Scheme of the binuclear manganese cluster and the localization

of the side-chains of Asp153and Asn153in the modelled structures of wild-type and D153N mutant forms of E coli agmatinase Average distances (in A˚)are indicated by the numbers For simplicity, other active site residues, including other metal ligands, are not indicated.

Fig 3 CD spectra of wild-type (solid line) and D153N mutant (dotted

line) E coli agmatinases The far-UV CD spectra were recorded at

22 C.

potential metal ligands are not substantially affected by the

mutation, as revealed by the conservation of the whole

topology of the active site, would explain the essentially

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

A C K N O W L E D G E M E N T S

This research was supported by Grant 2990049 from FONDECYT and

P.I 98.031.076-1.0 from the Direccio´n de Investigacio´n, Universidad de

Concepcio´n We are greateful to Dr Enrique Pe´rez Paya (Universidad

de Valencia, Espan˜a)for assistance with the CD spectra.

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