Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillus fumigatus

• Published in: The Journal of biological chemistry 2020-11, Vol.295 (45), p.15407-15417
• Main Authors: Hira, Daisuke, Onoue, Takuya, Oka, Takuji
• Format: Article
• Language: English
• Subjects: Aspergillus; Aspergillus fumigatus - cytology; Aspergillus fumigatus - metabolism; cell wall; Cell Wall - chemistry; Cell Wall - metabolism; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Glycobiology and Extracellular Matrices; glycosyltransferase; mannan; Mannans - biosynthesis; Mannans - chemistry; mannosyltransferase; Mannosyltransferases - chemistry; Mannosyltransferases - genetics; Mannosyltransferases - metabolism; molecular dynamics; Molecular Dynamics Simulation; protein structure
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA120.013742

Fungal cell walls and their biosynthetic enzymes are potential targets for novel antifungal agents. Recently, two mannosyltransferases, namely core-mannan synthases A (CmsA/Ktr4) and B (CmsB/Ktr7), were found to play roles in the core-mannan biosynthesis of fungal-type galactomannan. CmsA/Ktr4 is an α-(1→2)-mannosyltransferase responsible for α-(1→2)-mannan biosynthesis in fungal-type galactomannan, which covers the cell surface of Aspergillus fumigatus. Strains with disrupted cmsA/ktr4 have been shown to exhibit strongly suppressed hyphal elongation and conidiation alongside reduced virulence in a mouse model of invasive aspergillosis, indicating that CmsA/Ktr4 is a potential novel antifungal candidate. In this study we present the 3D structures of the soluble catalytic domain of CmsA/Ktr4, as determined by X-ray crystallography at a resolution of 1.95 Å, as well as the enzyme and Mn2+/GDP complex to 1.90 Å resolution. The CmsA/Ktr4 protein not only contains a highly conserved binding pocket for the donor substrate, GDP-mannose, but also has a unique broad cleft structure formed by its N- and C-terminal regions and is expected to recognize the acceptor substrate, a mannan chain. Based on these crystal structures, we also present a 3D structural model of the enzyme–substrate complex generated using docking and molecular dynamics simulations with α-Man-(1→6)-α-Man-(1→2)-α-Man-OMe as the model structure for the acceptor substrate. This predicted enzyme–substrate complex structure is also supported by findings from single amino acid substitution CmsA/Ktr4 mutants expressed in ΔcmsA/ktr4 A. fumigatus cells. Taken together, these results provide basic information for developing specific α-mannan biosynthesis inhibitors for use as pharmaceuticals and/or pesticides.