Tài liệu Báo cáo khoa học: Pyrimidine-specific ribonucleoside hydrolase from the archaeon Sulfolobus

Pyrimidine-specific ribonucleoside hydrolase from the

archaeon Sulfolobus solfataricus – biochemical

characterization and homology modeling

Marina Porcelli1,2, Luigi Concilio1

, Iolanda Peluso1

, Anna Marabotti3

, Angelo Facchiano3 and

Giovanna Cacciapuoti1

1 Dipartimento di Biochimica e Biofisica ‘F. Cedrangolo’, Seconda Universita` di Napoli, Italy

2 Consorzio Interuniversitario Biostrutture e Biosistemi ‘INBB’, Rome, Italy

3 Istituto di Scienze dell’Alimentazione del CNR, Avellino, Italy

Nucleoside hydrolases (NHs; EC 3.2.2.–) catalyze the

irreversible hydrolysis of the N-glycosidic bond of

b-ribonucleosides, forming ribose and the free purine

or pyrimidine base [1–3]. All characterized members

are metalloproteins with a unique central b-sheet

topology and a cluster of aspartate residues

(DXDXXXDD motif) at the N-terminus of the

enzyme [2–5].

In nature, a widespread distribution of NHs in dif￾ferent protozoa [6–11], bacteria [12–14], yeasts [15–17],

insects [18] and mesozoa [19] is observable. Genes con￾taining the characteristic NH structural motif have

been also found in plants [20,21], amphibians and

fishes [3].

Nucleoside hydrolases play a well-established

key role in the purine salvage pathway of parasitic

Keywords

homology modeling; hyperthermostability;

nucleoside hydrolase; nucleoside

metabolism; Sulfolobus solfataricus

Correspondence

M. Porcelli, Dipartimento di Biochimica e

Biofisica ‘F. Cedrangolo’, Seconda Universita`

di Napoli, Via Costantinopoli 16,

Napoli 80138, Italy

Fax: +39 081 5667519

Tel: +39 081 5667545

E-mail: [email protected]

(Received 23 November 2007, revised 11

February 2008, accepted 20 February 2008)

doi:10.1111/j.1742-4658.2008.06348.x

We report the characterization of the pyrimidine-specific ribonucleoside

hydrolase from the hyperthermophilic archaeon Sulfolobus solfataricus

(SsCU-NH). The gene SSO0505 encoding SsCU-NH was cloned and

expressed in Escherichia coli and the recombinant protein was purified to

homogeneity. SsCU-NH is a homotetramer of 140 kDa that recognizes

uridine and cytidine as substrates. SsCU-NH shares 34% sequence identity

with pyrimidine-specific nucleoside hydrolase from E. coli YeiK. The align￾ment of the amino acid sequences of SsCU-NH with nucleoside hydrolases

whose 3D structures have been solved indicates that the amino acid resi￾dues involved in the calcium- and ribose-binding sites are preserved.

SsCU-NH is highly thermophilic with an optimum temperature of 100 C

and is characterized by extreme thermodynamic stability (Tm = 106 C)

and kinetic stability (100% residual activity after 1 h incubation at 90 C).

Limited proteolysis indicated that the only proteolytic cleavage site is local￾ized in the C-terminal region and that the C-terminal peptide is necessary

for the integrity of the active site. The structure of the enzyme determined

by homology modeling provides insight into the proteolytic analyses as well

as into mechanisms of thermal stability. This is the first nucleoside hydro￾lase from Archaea.

Abbreviations

Cf, Crithidia fasciculata; CU-NH, pyrimidine-specific ribonucleoside hydrolases; Ec, Escherichia coli; IAG-NH, purine-specific inosine￾adenosine-guanosine nucleoside hydrolases; IG-NH, 6-oxo-purine-specific inosine-guanosine nucleoside hydrolases; IPTG, isopropyl thio-b￾D-galactoside; IU-NH, purine-nonspecific inosine-uridine nucleoside hydrolases; Lm, Leishmania major; MTA, 5¢-methylthioadenosine; MTAP,

5¢-methylthioadenosine phosphorylase; MTAPII, 5¢-methylthioadenosine phosphorylase II; MTI, methylthioinosine; NH, nucleoside hydrolase;

NP, nucleoside phosphorylase; PNP, purine nucleoside phosphorylase; PVDF, poly(vinylidene fluoride); Ss, Sulfolobus solfataricus;

Tv, Trypanosoma vivax.

1900 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS