Tài liệu Báo cáo Y học: The glucose-specific carrier of the Escherichia coli phosphotransferase

The glucose-specific carrier of the Escherichia coli phosphotransferase

system

Synthesis of selective inhibitors and inactivation studies

Luis Fernando Garcı´a-Alles, Vera Navdaeva, Simon Haenni and Bernhard Erni

Departement fu¨r Chemie und Biochemie, Universita¨t Bern, Freiestrasse 3, CH-3012, Bern, Switzerland

Thirteen glucose analogues bearing electrophilic groups

were synthesized (five of them for the first time) and screened

as inhibitors of the glucose transporter (EIIGlc) of the

Escherichia coli phosphoenolpyruvate–sugar phospho￾transferase system (PTS). 2¢,3¢-Epoxypropyl b-D-glucopyr￾anoside (3a) is an inhibitor and also a pseudosubstrate. Five

analogues are inhibitors of nonvectorial Glc phosphorylation

by EIIGlc but not pseudosubstrates. They are selective for

EIIGlc as demonstrated by comparison with EIIMan, another

Glc-specific but structurally different transporter. 3a is the

only analogue that inhibits EIIGlc by binding to the high￾affinity cytoplasmic binding site and also strongly inhibits

sugar uptake mediated by this transporter. The most potent

inhibitor in vitro, methyl 6,7-anhydro-D,L-glycero-a-D￾gluco-heptopyranoside (1d), preferentially interacts with the

low-affinity cytoplasmic site but only weakly inhibits Glc

uptake. Binding and/or phosphorylation from the cyto￾plasmic side of EIIGlc is more permissive than sugar binding

and/or translocation of substrates via the periplasmic site.

EIIGlc is rapidly inactivated by the 6-O-bromoacetyl esters of

methyl a-D-glucopyranoside (1a) and methyl a-D-manno￾pyranoside (1c), methyl 6-deoxy-6-isothiocyanato-a-D￾glucopyranoside (1e), b-D-glucopyranosyl isothiocyanate

(3c) and b-D-glucopyranosyl phenyl isothiocyanate (3d).

Phosphorylation of EIIGlc protects, indicating that inacti￾vation occurs by alkylation of Cys421. Glc does not protect,

but sensitizes EIIGlc for inactivation by 1e and 3d, which is

interpreted as the effect of glucose-induced conformational

changes in the dimeric transporter. Glc also sensitizes EIIGlc

for inactivation by 1a and 1c of uptake by starved cells. This

indicates that Cys421 which is located on the cytoplasmic

domain of EIIGlc becomes transiently accessible to substrate

analogues on the periplasmic side of the transporter.

Keywords: binding site; carbohydrate chemistry; cysteine;

glucose transporter; irreversible inhibitor.

Escherichia coli has two transporters for glucose, EIIGlc

(IIAGlc-IICBGlc) [1] and EIIMan (IIABMan-IICMan-IIDMan)

[2,3], which mediate uptake concomitant with phosphory￾lation of their substrates. The immediate source of high￾energy phosphate is the phosphoryl carrier protein HPr

which in turn is phosphorylated by phosphoenolpyruvate in

a reaction catalysed by enzyme I (EI). EI and HPr together

with the carbohydrate transporters (enzymes II, EIIsugar) of

diverse specificity and structure are components of the

bacterial phosphoenolpyruvate–sugar phosphotransferase

system (PTS) [4]. The PTS in addition comprises a number

of proteins that act as allosteric regulators of enzymes and/

or transcription factors.

The PTS transporters are homodimers, as indicated by

cross-linking, ultracentrifugation, gel filtration, interallelic

complementation and cryo-electron crystallography [5–9].

One protomer comprises three (or four) functional units,

IIA, IIB and IIC(IID), which occur either as protein

subunits or as domains in polypeptide chains. IIA and IIB

sequentially transfer phosphoryl groups from HPr to the

transported sugars. IIC contains the major determinants for

sugar recognition and translocation, as inferred from

binding studies [10] and the substrate selectivity of a

chimeric EIIGlcNAc/Glc [11]. EI, HPr and IIA are phos￾phorylated at His, whereas IIB domains are phosphorylated

at Cys421 in EIIGlc and at His175 in EIIMan. EIIGlc is

specific for Glc, but EIIMan has a broader substrate

specificity for Glc, Man, and other derivatives of Glc

altered at the C-2 carbon. Both transporters phosphorylate

their hexose substrates at OH-6. In spite of their overlapping

substrate specificity and analogous mechanism of action,

EIIGlc and EIIMan do not share amino-acid sequence

similarity, and, as judged by the known X-ray structures

of their cytoplasmic domains, also assume completely

different folds (for a review see [12]). The topology of the

membrane-spanning units IICGlc and IICMan-IIDMan are

also different, as judged by the characterization of protein

fusions between C-terminally truncated IIC(D) domains

with alkaline phosphatase and b-galactosidase [13,14].

Whereas the sites of EII phosphorylation are known and

easily recognized from the invariant amino-acid sequence

motifs, residues participating in sugar binding have not been

identified. Each protomer has been proposed to have a

sugar-binding site of its own with the two sites in the dimer

being distinguished by their different affinity for the

substrate [15]. Both sites are simultaneously accessible from

the cytoplasmic face. The IICBGlc subunits co-operate in so

Correspondence to L. F. Garcı´a Alles, Departement fu¨r Chemie und

Biochemie, Universita¨t Bern, Freiestrasse 3, CH-3012 Bern,

Schweiz. Fax: + 41 31 631 48 87, Tel.: + 41 31 631 37 92,

E-mail: [email protected]

Abbreviations: PTS, phosphoenolpyruvate–sugar phosphotransferase

system; aMGlc, methyl a-D-glucopyranoside; 2dGlc, 2-deoxy-D-glucose;

IC50, half inhibitory concentration; FC, flash chromatography.

(Received 9 June 2002, revised 16 August 2002,

accepted 21 August 2002)

Eur. J. Biochem. 269, 4969–4980 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03197.x