Molecular cloning and characterization of a cDNA encoding the N-acetyl-β-D-glucosaminidase homologue of Paracoccidioides brasiliensis

• Published in: Medical mycology (Oxford) 2004-06, Vol.42 (3), p.247-253
• Main Authors: Santos, Mônica O., Pereira, Maristela, Felipe, Maria Sueli S., Jesuino, Rosalia Santos A., Ulhoa, Cirano J., Soares, Renata De Bastos A., Soares, Celia Maria De A.
• Format: Article
• Language: English
• Subjects: 5' Untranslated Regions - genetics; Acetylglucosaminidase - genetics; Acetylglucosaminidase - isolation & purification; Amino Acid Sequence; Aspergillus nidulans; Aspergillus nidulans - genetics; Base Sequence; Binding Sites - genetics; Candida albicans; Candida albicans - genetics; Catalytic Domain - genetics; Cloning, Molecular; Codon, Initiator - genetics; Codon, Terminator - genetics; Conserved Sequence - genetics; DNA, Complementary - chemistry; DNA, Complementary - genetics; DNA, Complementary - isolation & purification; DNA, Fungal - chemistry; DNA, Fungal - genetics; DNA, Fungal - isolation & purification; Fungal Proteins - genetics; Fungal Proteins - physiology; Genes, Fungal - genetics; Genes, Fungal - physiology; Molecular Sequence Data; Molecular Weight; Open Reading Frames - genetics; Paracoccidioides - enzymology; Paracoccidioides - genetics; Paracoccidioides brasiliensis; Phylogeny; Protein Sorting Signals - genetics; RNA 3' Polyadenylation Signals - genetics; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Trichoderma - genetics; Trichoderma harzianum
• ISSN: 1369-3786; 1460-2709
• DOI: 10.1080/13693780310001644671

A cDNA encoding the N-acetyl-β-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized. The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with 'classic' NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans. In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.