Functional characterization of GDP-mannose pyrophosphorylase from Leptospira interrogans serovar Copenhageni

MATıAS D. ASENCION DIEZ; ANA DEMONTE; JORGE GIACOMELLI; SERGIO GARAY; DANIEL RODRÍGUES; BIRGIT HOFMANN; HANS-JUERGUEN HECHT; SERGIO A. GUERRERO; ALBERTO A. IGLESIAS

ARCHIVES OF MICROBIOLOGY

SPRINGER

Año: 2010 vol. 192 p. 103 - 114

0302-8933

Leptospira interrogans synthesizes a range of mannose-containing glycoconjugates relevant for its virulence. A prerequisite in the synthesis is the availability of the GDP-mannose, produced from mannose-1-phosphate and GTP in a reaction catalyzed by GDP-mannose pyrophosphorylase. The gene coding for a putative enzyme in L. interrogans was expressed in Escherichia coli BL21(DE3). The identity of this enzyme was confirmed by electrospray-mass spectroscopy, Edman sequencing and immunological assays. Gel filtration chromatography showed that the dimeric form of the enzyme is catalytically active and stable. The recombinant protein was characterized as a mannose-1-phosphate guanylyltransferase. S0.5 for the substrates were determined both in GDPmannose pyrophosphorolysis: 0.20 mM (GDP-mannose), 0.089 mM (PPi), and 0.47 mM; and in GDP-mannose synthesis: 0.24 mM (GTP), 0.063 mM (mannose-1-phosphate), and 0.45 mM (Mg2?). The enzyme was able to produce GDP-mannose, IDP-mannose, UDP-mannose and ADP-glucose. We obtained a structural model of the enzyme using as a template the crystal structure of mannose- 1-phosphate guanylyltransferase from Thermus thermophilus HB8. Binding of substrates and cofactor in the model agree with the pyrophosphorylases reaction mechanism. Our studies provide insights into the structure of a novel molecular target, which could be useful for detection of leptospirosis and for the development of antileptospiral drugs.