Tài liệu Báo cáo khoa học: Role of K22 and R120 in the covalent binding of the antibiotic fosfomycin

Role of K22 and R120 in the covalent binding of the antibiotic

fosfomycin and the substrate-induced conformational change in

UDP-N-acetylglucosamine enol pyruvyl transferase

Alison M. Thomas1,*, Cristian Ginj1,*, Ilian Jelesarov2

, Nikolaus Amrhein1 and Peter Macheroux1

1

Eidgeno¨ssische Technische Hochschule Zu¨rich, Institute of Plant Sciences, Department of Agricultural and Food Sciences and

Department of Biology, Zu¨rich, Switzerland; 2

Universita¨t Zu¨rich, Institute of Biochemistry, Zu¨rich, Switzerland

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA),

catalyzes the first step in the biosynthesis of peptidoglycan,

involving the transfer of the intact enolpyruvyl moiety from

phosphoenolpyruvate to the 3¢-hydroxyl group of UDP-N￾acetylglucosamine (UDPNAG). The enzyme is irreversibly

inhibited by the antibiotic fosfomycin. The inactivation is

caused by alkylation of a highly conserved cysteine residue

(C115) that participates in the binding of phos￾phoenolpyruvate. The three-dimensional structure of the

enzyme suggests that two residues may play a decisive role in

fosfomycin binding: K22 and R120. To investigate the role

of these residues, we have generated the K22V, K22E, K22R

and R120K single mutant proteins as well as the K22V/

R120K and K22V/R120V double mutant proteins. We

demonstrated that the K22R mutant protein behaves simi￾larly to wild-type enzyme, whereas the K22E mutant protein

failed to form the covalent adduct. On the other hand, the

K22V mutant protein requires the presence of UDPNAG

for the formation of the adduct indicating that UDPNAG

plays a crucial role in the organization of productive inter￾actions in the active site. This model receives strong support

from heat capacity changes observed for the K22V/R120K

and R120K mutant proteins: in both mutant proteins, the

heat capacity changes are markedly reduced indicating

that their ability to form a closed protein conformation is

impeded due to the R120K exchange.

Keywords: transferase; fosfomycin; antibiotic; mutagenesis;

protein conformation.

A rigid cell wall is essential for the survival of most bacteria.

Compounds that interfere with cell wall biosynthesis or

function, such as b-lactams, are powerful antibiotics and the

bacterial enzymes involved in cell wall biosynthesis are

attractive targets for the development of new drugs [1]. The

biosynthesis of the cell wall component peptidoglycan (or

murein) commences with the transfer of the intact enolpyr￾uvyl moiety of phosphoenolpyruvate to the 3¢-hydroxyl

group of UDP-N-acetylglucosamine (UDPNAG) [2]. This

reaction, catalysed by UDP-N-acetylglucosamine enol￾pyruvyl transferase (MurA), leads to the generation of

UDP-N-acetylenolpyruvylglucosamine (Scheme 1A). The

naturally occurring antibiotic fosfomycin, produced by some

Streptomyces and Pseudomonas species [3–5], irreversibly

inhibits MurA activity by alkylating the thiol group of a

catalytically important cysteine residue, C115 (Scheme 1B)

[6].

The rate of MurA inactivation by fosfomycin is increased

considerably in the presence of UDPNAG [7]. This accel￾erating effect is not due to a change in the reactivity of the

thiol group, as the pKa of the thiol group is not affected by

UDPNAG binding [8]. Crystallographic studies have shown

that MurA is subject to a large conformational change upon

binding of UDPNAG and fosfomycin or UDPNAG and

(Z)-3-fluorophosphoenolpyruvate, respectively, to the free,

unliganded enzyme [9–11] (Fig. 1). In the unliganded form,

the active site of MurA is readily accessible (
open confor￾mation) whereas in the liganded form (
closed conforma￾tion) a loop in the upper domain forms a lid on the active

site, thereby shielding the ligands from solvent and gener￾ating a compact structure. This loop movement places the

reactive C115 closer to fosfomycin or (Z)-3-fluorophos￾phoenolpyruvate in the active site (Fig. 1). Hence it can be

assumed that fosfomycin and the thiol group of C115 are

optimally positioned in the closed conformation, so that the

nucleophilic attack of the thiol group is facilitated.

In a recently initiated site-directed mutagenesis program,

we have discovered that replacement of K22 leads to a more

than 300-fold decrease in enzymatic activity [12]. Using

isothermal titration calorimetry (ITC), fosfomycin binding

was detected for the conservative mutation K22R in the

presence of UDPNAG while the K22V and K22E mutant

proteins appeared to have lost this ability completely [12].

According to the three-dimensional structure of MurA [11],

Correspondence to P. Macheroux, Graz University of Technology,

Institute of Biochemistry, Petersgasse 12/II, A-8010 Graz, Austria.

Fax: + 43 316 873 6952, Tel.: + 43 316 873 6450,

E-mail: [email protected]

Abbreviations: fosfomycin, (1R,2S)-1,2-epoxypropylphosphonic acid;

glyphosate, N-(phosphonomethyl)-glycine; ITC, isothermal titration

calorimetry; MurA, UDP-N-acetylglucosamine enolpyruvyl trans￾ferase; TPCK, L-(tosylamido-2-phenyl) ethyl chloromethyl ketone;

UDPNAG, UDP-N-acetylglucosamine; DCp, heat capacity change;

DG, free energy change; DH, enthalpy change; DS, entropy change.

*Note: The first two authors contributed equally to this work.

(Received 16 February 2004, revised 26 April 2004,

accepted 30 April 2004)

Eur. J. Biochem. 271, 2682–2690 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04196.x