Molecular cloning and analysis of the NAG1 cDNA coding for glucosamine-6-phosphate deaminase from Candida albicans.
Candida albicans and other pathogenic Candida species can use N-acetylglucosamine as a sole carbon source for growth. GlcNAc induces the enzymes of GlcNAc catabolic pathway; besides, under certain conditions, GlcNAc also induces a change from the yeast to germ tube morphology. Glucosamine-6-phosphate deaminase (EC 5.3.1.10) is the terminal enzyme of the GlcNAc catabolic pathway. We have purified the deaminase from C. albicans and studied its characteristics. The size of the deaminase estimated from SDS-polyacrylamide gel electrophoresis is 28 kDa. N-Acetylglucosamine 6-phosphate, an allosteric activator of the Escherichia coli deaminase, has no effect on the activity of the C. albicans enzyme. The deaminase is induced over 100-fold by GlcNAc and its level is about 0.3-0.5% of the proteins in crude extract. Three cDNA clones were obtained from a lambda gt11 expression library by immunoscreening with deaminase antiserum. C. albicans genomic DNA blot hybridization revealed that the NAG1 gene, encoding the glucosamine-6-phosphate deaminase, is present in a single copy. Hybrid-selected translation and immunoprecipitation experiments revealed that the purified deaminase and the protein encoded by the clones were similar in size and in their antigenicity. DNA sequencing revealed that the largest cDNA clone contained the complete open reading frame, which can code for a 27.5-kDa protein. The NH2-terminal sequence (35 residues) determined from the purified deaminase was identical to the sequence of the deduced protein. The Nag1 protein has about 47% identity with the sequence of the E. coli glucosamine-6-phosphate deaminase. Furthermore, RNA blot hybridization showed that GlcNAc induces the expression of NAG1 gene.
Natarajan
K
K
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Datta
A
A
D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D000906:Antibodies / Q000302:isolation & purification; D001483:Base Sequence; D015139:Blotting, Southern; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics*; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D005800:Genes, Fungal; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D004271:DNA, Fungal; D007202:Indicators and Reagents; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011061:Poly A; D012313:RNA; C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases