The presence of β-mannosides in their cell walls confers specific features on the pathogenic yeasts Candida albicans and Candida glabrata compared with non-pathogenic yeasts. In the present study, we investigated the enzymatic properties of Bmt1 (β-mannosyltransferase 1), a member of the recently identified β-mannosyltransferase family, from C. albicans. A recombinant soluble enzyme lacking the N-terminal region was expressed as a secreted protein from the methylotrophic yeast Pichia pastoris. In parallel, functionalized natural oligosaccharides isolated from Saccharomyces cerevisiae and a C. albicans mutant strain, as well as synthetic α-oligomannosides, were prepared and used as potential acceptor substrates. Bmt1p preferentially utilizes substrates containing linear chains of α-1,2-linked mannotriose or mannotetraose. The recombinant enzyme consecuti-vely transfers two mannosyl units on to these acceptors, leading to the production of α-mannosidase-resistant oligomannosides. NMR experiments further confirmed the presence of a terminal βMan (β-1,2-linked mannose) unit in the first enzyme product. In the future, a better understanding of specific β-1,2-mannosyltransferase molecular requirements will help the design of new potential antifungal drugs.
- cell wall
Abbreviations: Bmt, β-mannosyltransferase; CMC, critical micellar concentration; ConA, concanavalin A; DDM, n-dodecyl-β-D-maltoside; DLS, dynamic light scattering; EndoH, endoglycosidase H; GT, glycosyltransferase; HM, heptyl mannose; LDAO, N,N-dimethyldodecylamine-N-oxide; αMan, α-linked mannose; βMan, β-1,2-linked mannose; NP-FL-HPLC, normal-phase HPLC with fluorimetric detection; PA, pyridylamino; PLM, phospholipomannan; PNGase F, peptide N-glycosidase F; PPM, phosphopeptidomannan
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