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. 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.

The ADP‐glucose pyrophosphorylase from Streptococcus mutans provides evidence for the regulation of polysaccharide biosynthesis in Firmicutes

“…It has been determined that pyrophosphorylases are relatively specific for their respective substrates, although some exceptions have been reported. For example, dTTP is a poor substrate for UDP‐Glc PPase from some bacteria (Weissborn et al ., ; Bosco et al ., ) and for GDP‐mannose (GDP‐Man) PPase from Mycobacterium tuberculosis and Leptospira interrogans are promiscuous regarding nucleotide tri‐phosphates (NTPs) (Ning and Elbein, ; Asencion Diez et al ., ). We explored the possibility for using different NTPs (UTP, ITP, GTP, dTTP) as substrates alternative to ATP by both S. mutans GlgC and GlgC/GlgD.…”

A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

“…For example, dTTP is a substrate in some bacterial UDP-Glc PPases [8,11,32], whereas GDP-mannose (GDP-Man) PPases from M. tuberculosis and from Leptospira interrogans exhibit promiscuity in the use of the nucleotide triphosphate (NTP) substrate [33,34]. Different NTPs (ATP; UTP; ITP; GTP; CTP; dTTP) at 0.1, 0.5 and 2.5 mM final concentration were assayed as alternative substrates for Smu GalU and Smu GalU-Δ294 Eco GlgC.…”

DoGMP1 from Dendrobium officinale contributes to mannose content of water-soluble polysaccharides and plays a role in salt stress response

“…GMP catalyzes the reaction from mannose-1-phosphate to GDP-mannose and uses cofactor Mg 2+ as a key switch for effective and continuous enzyme production 42 43 . Two types of GMP genes, namely GDP - mannose pyrophosphorylase A ( GMPA ) and GDP - mannose pyrophosphorylase B ( GMPB ), were found in fungus ( Saccharomyces cerevisiae ) 44 45 , pig ( Sus scrofa ) 46 , humans ( Homo sapiens ) 29 and a higher plant, rice 10 .…”