D-Arabinose Methabolism: Characterization of Bifunctional Arabinokinase/ Pyrophosphorylase of Leishmania major

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Abstract


In this work we describe an unusual enzyme from Leishmania major (Arabinokinase/Pyrophosphorylase) that catalyzes the synthesis of GDP-Darabinopyranose (GDP-D-Ara p) via a D-arabinose-1-phosphate intermediate in the presence of ATP and GTP. Our data indicate GDP-D-Ara p transport in vivo by the LPG2 multispecific nucleotide sugar transporter into the Leishmania Golgi apparatus, in which it can be used by glycosyltransferases as a donor substrate for glycosylation.

Introd uction It is known that both bacteria (Bacteriodes) and plants (Arabidopsis) can synthesize GDP-L-Fuc from L-fucose (L-Fuc) via intermediate L-fucose-1-phosphate using a bifunctional enzyme L-fucokinase/GDP-L-fucose pyrophosphorylase [3, 4]. Since D-Arap and L-Fuc are structurally similar, it makes sense to assume that biosynthesis of GDP-D-Arap in Leishmania can occur through a mechanism similar to that of GDPLFuc biosynthesis in other species. To check this hypothesis, the L.major genome was evaluated for open reading frames homologous to fucokinase and GDP-L-fucose pyrophosphorylase gene sequences. As a result, two near-identical genes (lmjF16.0440 and lmjF16.0480) were found as possessing high homology with the Bacteriodes fragilis fkp and Arabidopsis thaliana at1g01220 genes, both encoding L-fucokinase/GDPLfucose pyrophosphorylase [3, 4]. The open reading frames lmjF16.0440 and lmjF16.0480 correspond to putative polypeptides composed of 1,187 aminoacid residues with a calculated molecular mass of 126.5 kDa. The only difference (three aminoacid residues) between these two proteins is the segment 196–199, namely Phe-lnAsn-is in LmjF16.0480 and Leu-lnAspTyr in LmjF16.0440. The N-terminal sequences of these proteins contain a highly conserved segment Val100–Lys117 that closely resembles the conserved pyrophosphorylase motif Lys(X)2GlyXThrXMet(X)4Lys [5].

N M Novozhilova

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS

Email: nnovogilov@gmail.com

N V Bovin

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS

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Copyright (c) 2009 Novozhilova N.M., Bovin N.V.

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