Functional characterization of GDP-mannose pyrophosphorylase from Leptospira interrogans serovar Copenhageni.
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. S (0.5) 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 (Mg(2+)). 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.
Asención Diez
Matías D
MD
Laboratorio de Bioquímica Microbiana, Universidad Nacional del Litoral, Santa Fe, Argentina.
Demonte
Ana
A
Giacomelli
Jorge
J
Garay
Sergio
S
Rodrígues
Daniel
D
Hofmann
Birgit
B
Hecht
Hans-Juerguen
HJ
Guerrero
Sergio A
SA
Iglesias
Alberto A
AA
D000245:Adenosine Diphosphate Glucose / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002850:Chromatography, Gel; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D007921:Leptospira interrogans / Q000201:enzymology*; D008360:Mannosephosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D014539:Uridine Diphosphate Sugars / Q000378:metabolism
D001426:Bacterial Proteins; D008360:Mannosephosphates; D014539:Uridine Diphosphate Sugars; C042056:uridine diphosphate mannose; D000245:Adenosine Diphosphate Glucose; C047217:mannose 1-phosphate; D006155:Guanosine Diphosphate Mannose; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase