Glucosamine Fructose 6‐phosphate Aminotransferase (GFAT) from Plasmodium berghei: a clue for novel therapeutic perspectives?

Abstract

Glucosamine Fructose 6‐phosphate Aminotransferase (GFAT) is the first enzyme involved in hexosamine pathway, both in Eukaryotes and Prokaryotes. The hexosamine pathway leads to the formation of UDP‐N‐acetyl‐glucosamine (UDP‐GlcNAc). GlcNAc is widely used in most cellular organisms, from prokaryotes to eukaryotes and it essential for glycoproteins, glycolipids, proteoglycans and polysaccharides formation.GFAT (identified as GmlS in Bacteria) displays two domains: a N‐terminal glutaminase domain and a C‐terminal isomerase one. The C‐domain catalyzes the conversion of Fru6P to Glucosamine‐6P (GclN‐6P). Then, an NH4+ molecule derived from the glutaminase activity of the N‐domain is shuttled to the C‐term region via a hydrophobic channel that helps to prevent its loss to the solvent and then transferred to the sugar.Using site‐directed mutagenesis the Cys1 residue has been demonstrated to be catalytically essential and it has been suggested to act as a nucleophile to attack the amide carbon of the glutamine substrate; moreover, evidences indicated that the free N‐terminal Cys1 amino group is also essential for catalysis, in agreement with other members of the N‐terminal nucleophile hydrolase family. Indeed, all GFAT (and bacterial GlmS) so far characterized, after processing by Met aminopeptidase, display an N‐terminal Cys.However, sequence analyses in protein databases have revealed that a group of protozoan GFAT does not follow this rule, displaying long N‐terminal extensions, thus challenging the proposed catalytic mechanism. These N‐terminal extensions do not have apparently a clear biological significance in the analyzed organisms: they are highly variable and do not display known signal sequences for targeting in sub‐cellular compartments. Moreover, these GFAT sequences also lack an 40–70 aminoacid insertion, present in the typical eukaryotic enzymes, but also absent in the bacterial GlmS. This insertion is involved in feed‐back regulation of enzyme activity by UDP‐GlcNAc, the final product of the pathway.To better understand the role of the N‐terminal extension in some protozoan GFAT, we have expressed Plasmodium berghei GFAT as wild type form and as a truncated form exposing the N‐terminal free Cys1. Enzymatic characterization has revealed that, despite the presence of the N‐terminal extension, wt GFAT has a higher glutaminase activity compared to the truncated form. These findings pose question about the proposed catalytic mechanism for this class of GFAT.Studies are in progress to identify the functional role of GFAT N‐terminal extension and the differences in the catalytic mechanism, compared to human GFAT. Since extension is present in important human protozoan pathogens, such as Apicomplexa (i.e. Plasmodium, Toxoplasma), Heterokonta (i.e. Blastocystis) and Amoeboza (i.e. Entamoeba, Acanhtamoeba), these studies could represent a starting point for the development of more specific inhibitors for therapeutic applications.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.