The VIG9 gene products from the human pathogenic fungi Candida albicans and Candida glabrata encode GDP-mannose pyrophosphorylase

We have identified two genomic DNA fragments from the human pathogenic fungi, Candida albicans (CaVIG9) and Candida glabrata (CgVIG9) that encode GDP-mannose pyrophosphorylase, a key enzyme for protein glycosylation. The VIG9 homologues of CaVIG9 and CgVIG9 complement an identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae. The nucleotide sequences of the ORFs, which are 83 and 90% identical to that of the ScVIG9 protein, respectively, showed a predicted gene product homologous to S. cerevisiae GDP-mannose pyrophosphorylase. We examined the enzyme activity of a glutathione S-transferase fusion of each VIG9 gene to synthesize GDP mannose in the cell extracts of a heterologous Escherichia coli expression system. We also developed a method for detecting the enzyme activity using a non-radioactive substrate that would be applicable to high throughput screening. (C) 2000 Elsevier Science B.V. All rights reserved.