Functional Characterization of GDP-mannose Pyrophosphorylase from Leptospira interrogans serovar Copenhageni

M.D. ASENCIÓN DIEZ; A.M. DEMONTE; J. GIACOMELLI; S. GARAY; D. RODRIGUES; B. HOFMANN; H-J. HECHT; S.A. GUERRERO; A.A. IGLESIAS

ARCHIVES OF MICROBIOLOGY

SPRINGER

Lugar: New York; 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 coliBL21(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 wascharacterized 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 theenzyme 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.