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

Date Published: October , 2009

Publisher: A.I. Gordeyev

Author(s): N.M. Novozhilova, N.V. Bovin.

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Abstract

In this work we describe an unusual enzyme from Leishmania major (Arabinokinase/Pyrophosphorylase) that catalyzes the synthesis of GDP-D-arabinopyranose (GDP-D-Arap) via a D-arabinose-1-phosphate intermediate in the presence of ATP and GTP. Our data indicate GDP-D-Arap 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.

Partial Text

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 GDP-L-Fuc 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/GDP-L-fucose pyrophosphorylase [3, 4].

The lmjF16.0440 and lmjF16.0480 genes were cloned in the pET16b plasmid vector for further examination of the activities of the putative enzymes. The recombinant LmjF16.0440 and LmjF16.0480 were expressed in E.coli BL21 (DE3)-RIPL cells and tested for enzyme activity (data are shown on Fig. 1). LmjF16.0480 catalyzed the formation of D-Arap-phosphate and GDP-D-Arap, whereas LmjF16.0440 only synthesized D-Arap-phosphate. Reaction products were not observed when the reaction mixture contained only nonspecific proteins extracted from the E.coli cells transformed with an empty pET16b vector. This information allows to conclude that the product of the lmjF16.0480 gene really acts as a bifunctional arabinokinase/pyrophosphorylase; that is, it exhibits both D-arabinokinase and GDP-D-Arap-pyrophosphorylase activity, whereas the recombinant LmjF16.0440 protein only exhibits D-arabinokinase activity.

In most cases, activated carbohydrates are synthesized in cytoplasm and subsequently transported into the Golgi lumen, in which they are utilized by corresponding glycosyltransferases as substrate donors in glycosylation reactions. Earlier, the LPG2 protein found in the Leishmania Golgi apparatus membrane was classified as a multispecific transporter that can transport not only GDP-Man, but also GDP-D-Arap and GDP-L-Fuc [7]. To make sure that LPG2 is the only transporter responsible for GDP-D-Arap transport into the Golgi apparatus, we grew the cells of wild-type L. major and its lpg2 knockout mutant (L. major lpg2-/-) in the presence of [3H]-arabinose and determined [3H]Ara incorporated into the cell surface glycoconjugates (fig. 2). One can see from the presented data that the lpg2 knockout cells do not incorporate [3H]-arabinose. The absence of arabinose in lipophosphoglycan of L. major lpg2-/- is easy to explain, because the loss of LPG2 leads to cancellation of GDP-Man transport into the Golgi apparatus with the corresponding termination of the synthesis of the carbohydrate moiety of the molecule [9]. Thus, even in the presence of GDP-Arap transported into the Golgi lumen by another protein, arabinosyltransferases had no acceptor site for arabinose transfer to lipophosphoglycan. There is no telling this about glycosylinositolphospholipids. Glycosylation of these molecules occurs through another biosynthetic pathway with dolichol-phosphate mannose as a donor [10], so the absence of arabinose residues in glycosylinositolphospholipid molecules of L. major lpg2-/- can be explained by cancellation of GDP-Arap transport into the Golgi apparatus in the cells devoid of lpg2 gene.

The study was supported by the Program of Presidium of Russian Academy of Sciences “Molecular and Cell Biology.” The authors are grateful to Prof. H. Guo (University of Washington, USA) for assistance in identification and cloning of Leishmania major and human genes and to Prof. S. Turco (University of Kentucky, USA) for fruitful discussions.

 

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