Biochemical characterization of Candida albicans alpha-glucosidase I heterologously expressed in Escherichia coli.
Protein glycosylation is one of the most common post-translational modifications present in the eukaryotic cell. The N-linked glycosylation is a biosynthetic pathway where an oligosaccharide is added to asparagine residues within the endoplasmic reticulum. Upon addition of the N-linked glycan to nascent proteins, alpha-glucosidase I removes the outermost alpha1,2-glucose unit from the N-linked core Glc(3)Man(9)GlcNAc(2). We have previously demonstrated that the endoplasmic reticulum α-glucosidase I is required for normal cell wall composition, and virulence of the human pathogen Candida albicans. In spite of the importance of this enzyme for normal cell biology, little is known about its structure and the amino acids participating in enzyme catalysis. Here, a DNA fragment corresponding to the 3'-end fragment of C. albicans CWH41, the encoding gene for α-glucosidase I, was expressed in a bacterial system and the recombinant peptide showed alpha-glucosidase activity, despite lacking 419 amino acids from the N-terminal end. The biochemical characterisation of the recombinant enzyme showed that presence of hydroxyl groups at carbons 3 and 6, and orientation of hydroxyl moiety at C-2 are important for glucose recognition. Additionally, results suggest that cysteine rather than histidine residues are involved in the catalysis by the recombinant enzyme.
Frade-Pérez
María D
MD
Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato Gto. CP 36000, Mexico.
Hernández-Cervantes
Arturo
A
Flores-Carreón
Arturo
A
Mora-Montes
Héctor M
HM
D000595:Amino Acid Sequence; D002176:Candida albicans / Q000145:classification / Q000201:enzymology*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D006031:Glycosylation; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C042059:glucosidase I; D000520:alpha-Glucosidases