Abstract

Glucosamine-6-phosphate (GlcN-6-P) synthase, known also as L-Glutamine: D-fructose-6-phosphate amidotransferase, catalyzes the first committed step in the pathway leading to the ultimate formation of UDP-GlcNAc. The final product of this pathway is an activated precursor of numerous macromolecules containing amino sugars, including chitin and mannproteins in fungi, peptidoglycan and lipopolysaccharides in bacteria, and glycoproteins in mammals. The reaction catalyzed by the enzyme is conversion of fructose-6-phosphate into glucosamine-6-phosphate with L-glutamine as a nitrogen donor. The important differences between prokaryotic and eukaryotic forms of the enzyme include a substantially larger size of the latter and sensitivity of the latter but not the former to the feedback inhibition by uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc). The enzyme has been proposed as a promising target in antifungal chemotherapy as well as for the treatment of insulin-independent diabetes mellitus. Our studies have been focused on GlcN-6-P synthase from human pathogenic fungi Candida albicans coded by the GFA1 gene. We were able to demonstrate a crystal structure of the isomerase domain (ISOM) of C. albicans GlcN-6-P synthase in complex with UDP-GlcNAc [2]. Now we report results of our further studies involving the site-directed mutagenesis of GFA1, leading to the following substitutions of amino acid residues located at the putative UDP-GlcNAc binding site in the gene products: Gly474Leu, Thr487Ile, Gly490Leu and His492Phe. Muteins obtaned at consecutive steps of mutagenesis were isolated and characterized. The final product of mutagenesis was completely insensitive to inhibition by UDP-GlcNAc, while sensitivities of the intermediate products were diminished. Conclusions concerning the probable role of UDP-GlcNAc binding for enzyme structure and catalytic properties are presented.

Authors (3)