title: Catalytic properties of Phr family members of cell wall glucan remodeling enzymes: implications for the adaptation of Candida albicans to ambient pH.
abstract: Fungal wall formation is a dynamic process involving several categories of enzymes. The GH72 family of β(1,3)-glucanosyltransferases is essential for the determination of cell shape, for cell integrity and for virulence in pathogenic fungi. Candida albicans has five GH72 genes: PHR1 and PHR2 are pH dependent, the first being expressed at pH ≥ 6 and repressed at lower pH and the second regulated in the opposite manner, PGA4 is transcribed independently of pH whereas PHR3 and PGA5 have low expression levels. To characterize the catalytic properties of Phr1p-2p and probe the activity of Pga4p, we heterologously expressed these proteins and used a fluorescent assay based on the transfer of oligosaccharyl units from a donor to a sulforhodamine-labeled acceptor. Phr1p-2p used exclusively β-1,3-glucan or cell wall glucan as donor and laminarin-derived oligosaccharides as acceptor. The acceptor efficiency increased with the length of the oligosaccharide. The temperature optimum was 30°C. The pH optimum was 5.8 for Phr1p and 3 for Phr2p. Overall, adaptation to pH of C. albicans appears to involve a fine interplay among the pH-dependent activity of Phr1p and Phr2p, the pH-regulated expression of their genes and protein stability. Unexpectedly, Pga4p was inactive suggesting that it turned into a structural mannoprotein.
authors.affiliation: Institute of Chemistry, Center for Glycomics, Department of Glycobiology, Slovak Academy of Sciences, 84538 Bratislava, Slovakia.
authors.forename: Kristína
authors.initials: K
authors.lastname: Kováčová
authors.affiliation: Università degli Studi di Milano, Dipartimento di Bioscienze, Via Celoria 26, 20133 Milano, Italy.
authors.forename: Genny
authors.initials: G
authors.lastname: Degani
authors.affiliation: Institute of Chemistry, Center for Glycomics, Department of Glycobiology, Slovak Academy of Sciences, 84538 Bratislava, Slovakia.
authors.forename: Eva
authors.initials: E
authors.lastname: Stratilová
authors.affiliation: Institute of Chemistry, Center for Glycomics, Department of Glycobiology, Slovak Academy of Sciences, 84538 Bratislava, Slovakia.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Farkaš
authors.affiliation: Università degli Studi di Milano, Dipartimento di Bioscienze, Via Celoria 26, 20133 Milano, Italy Laura.Popolo@unimi.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Popolo
mesh_terms: D000222:Adaptation, Physiological; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D002473:Cell Wall / Q000201:enzymology* / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism*; D016695:Glycosyltransferases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D005936:Glucans; D011994:Recombinant Proteins; D016695:Glycosyltransferases
keywords: Pichia pastoris expression; family GH72; fungal pathogens; glucanosyltransferases; morphogenesis; pH adaptation