Engineering Candida albicans glucosamine‐6‐phosphate synthase for efficient enzyme purification

Abstract

Rationally designed muteins of Candida albicans glucosamine-6-phosphate synthase, an enzyme known as a promising target for antifungal chemotherapy, were constructed, overexpressed in Escherichia coli and purified to near homogeneity. To facilitate and to optimize the purification of the enzyme, three recombinant versions containing internal oligoHis fragments were constructed: (i) by substituting residues 343-348 of the interdomain undecapeptide linker with hexaHis, (ii) by replacing solvent-exposed residues 655-660 of the isomerase domain with hexaHis, and (iii) by replacing amino acids at positions 568 and 569 with His residues to generate the three-dimensional hexaHis microdomain in the enzyme quaternary structure. The resulting constructs were effectively purified to near homogeneity by rapid, one-step immobilized metal-ion affinity chromatography and demonstrated activity and catalytic properties comparable with that of the wild-type enzyme. The construct containing the 655-660 hexaHis insert was found to be a homodimeric protein, which is the first reported example of such quaternary structure of glucosamine-6-phosphate synthase of eukaryotic origin.

Synthesis and biological activity of novel ester derivatives of N3-(4-metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid containing amide and keto function as inhibitors of glucosamine-6-phosphate synthase

“…Ester conjugates 4-10 have been obtained from the different hydroxyamides (which were previously synthesized from ethyl L-lactate and methyl glycolate), using combination of DCC and DMAP as a coupling agent. 12 The Boc-protected compounds All of the obtained FMDP analogues were tested as inhibitors of purified GlcN-6-P synthase (overproduced by Escherichia coli) 13 and their IC 50 values are presented in the Table 1.The ability of compounds 11-19 to inhibit this enzyme was measured by determining the concentration of inhibitor causing 50% inhibition of enzyme.…”