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

Abstract

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 GDP-mannose 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 anti-leptospiral drugs.

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Acknowledgments

This work was supported by grants from ANPCyT (PICTO′04 15-22427; PICTO′05 05-13469), CONICET (PIP 112-2008-01-02519), and UNL (CAI + D 2006, CAI + D 2009 Orientados & Redes). DER, SAG and AAI are investigator career members from CONICET. AAI is a Fellow from The John Simon Guggenheim Memorial Foundation.

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Correspondence to Alberto A. Iglesias.

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Communicated by Sebastian Suerbaum.

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Asención Diez, M.D., Demonte, A., Giacomelli, J. et al. Functional characterization of GDP-mannose pyrophosphorylase from Leptospira interrogans serovar Copenhageni. Arch Microbiol 192, 103–114 (2010). https://doi.org/10.1007/s00203-009-0534-3

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Keywords

  • Leptospira interrogans
  • Mannose metabolism
  • Pyrophosphorylase