Tài liệu Báo cáo khoa học: Structure of peptidase T from Salmonella typhimurium doc

Structure of peptidase T from Salmonella typhimurium

Kjell HaÊ kansson* and Charles G. Miller

Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

The structure of peptidase T, or tripeptidase, was deter￾mined by multiple wavelength anomalous dispersion

(MAD) methodology and re®ned to 2.4 AÊ resolution. Pep￾tidase T comprises two domains; a catalytic domain with an

active site containing two metal ions, and a smaller domain

formed through a long insertion into the catalytic domain.

The two metal ions, presumably zinc, are separated by 3.3 AÊ ,

and are coordinated by ®ve carboxylate and histidine

ligands. The molecular surface of the active site is negatively

charged. Peptidase T has the same basic fold as carboxy￾peptidase G2. When the structures of the two enzymes are

superimposed, a number of homologous residues, not evi￾dent from the sequence alone, could be identi®ed. Com￾parison of the active sites of peptidase T, carboxypeptidase

G2, Aeromonas proteolytica aminopeptidase, carboxypepti￾dase A and leucine aminopeptidase reveals a common

structural framework with interesting similarities and

di€erences in the active sites and in the zinc coordination.

A putative binding site for the C-terminal end of the

tripeptide substrate was found at a peptidase T speci®c

®ngerprint sequence motif.

Keywords: tripeptidase; aminotripeptidase; metallopep￾tidase; X-ray crystallography; MAD.

Escherichia coli and Salmonella typhimurium express several

intracellular enzymes capable of hydrolyzing peptides [1].

Many of these enzymes have been shown to function in the

degradation of intracellular proteins and in the catabolism

of exogenously supplied peptides [2]. One of these enzymes,

peptidase T, or tripeptidase (EC 3.4.11.-) is a 409 amino￾acid metalloenzyme that hydrolyzes tripeptides at their

N-termini [3,4]. The enzymatic activity of theS. typhimurium

enzyme is both speci®c and unusual; dipeptides, tetrapep￾tides or tripeptides with blocked N-termini are not cleaved.

S. typhimurium peptidase T expression is regulated by

FNR, a transcriptional activator that responds to anaero￾biosis [4,5]. The aerobic expression level of peptidase T is

not suf®cient to allow this enzyme to contribute to the

utilization of exogenously supplied peptides as amino acid

sources [3]. Under anaerobic conditions, however, the pepT

gene is induced, leading to levels of peptidase T that allow it

to participate in the catabolism of tripeptides [5,6]. It has

been speculated that this pattern of regulation may

contribute to the anaerobic utilization of amino acids as

energy sources [1].

A 45-amino-acid region of peptidase T displays similarity

to a short region in Pseudomonas sp. strain RS-16

carboxypeptidase G2 (CG2), peptidase D, and alkaline

phosphatase isozyme conversion peptidase (Iap) [4]. This

region of similarity contains two of the ®ve ligands that

coordinate the two zinc ions in the active site of CG2, for

which the three-dimensional structure is known [7]. Pepti￾dase T has therefore been classi®ed into the M20 family of

proteases/peptidases [8]. A third zinc ligand, a histidine, can

be recognized as part of a HXDT motif [9], which is

conserved in both peptidase T and CG2. While these data

indicate that peptidase T is evolutionarily related to CG2,

the lack of clear homology outside these regions, and the

unique tripeptidase speci®city of peptidase T, suggested that

the structure of the two enzymes would in part differ. We

report the three-dimensional structure of S. typhimurium

peptidase T solved by multiple wavelength anomalous

dispersion (MAD) methodology and re®ned to 2.4-AÊ

resolution.

MATERIALS AND METHODS

Crystallization and data collection

Selenomethionine His-tagged peptidase T was expressed in

strain TN5619, puri®ed, crystallized from ammonium

sulfate solutions at pH 7.5 and ¯ash frozen in 50% sucrose

as previously described [10]. Crystals belong to space group

C2 with a ˆ 132.4 AÊ , b ˆ 46.0 AÊ , c ˆ 96.6 AÊ , b ˆ

116.1 AÊ . Data were collected at 100 K at NSLS beam

station X4A (Brookhaven, NY, USA) at four different

wavelengths, and processed with DENZO, SCALEPACK and the

CCP4 program suite [11,12].

Structure solution and re®nement

The structure was solved by MAD methodology using 15

selenium atoms, four wavelengths and data to 2.8 AÊ .

Determination of selenium positions, phase and electron

density calculations and model re®nement were performed

with the CNS program package [13]. The model was built

manually and displayed using the graphics program O [14].

The model was re®ned against one of the data sets processed

Correspondence to C. G. Miller, Department of Microbiology, Uni￾versity of Illinois at Urbana-Champaign, B103 CLSL, 601 S. Good￾win Avenue, Urbana, Illinois 61801, USA. Fax: + 217 244 6697,

Tel.: + 217 244 8418, E-mail: [email protected]

Abbreviations: MAD, multiple wavelength anomalous dispersion;

CG2, carboxypeptidase G2; APP, Aeromonas proteolytica amino￾peptidase; LAP, leucine aminopeptidase; CPA, carboxypeptidase A.

*Present address: Laboratory of Cellular and Molecular Physiology,

August Krogh Institute, Universitetsparken 13, DK 2100, Kbh é,

Denmark.

(Received 3 September 2001, revised 6 November 2001, accepted 8

November 2001)

Eur. J. Biochem. 269, 443±450 (2002) Ó FEBS 2002