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 fung i, 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 glycosyla tion-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. ce revisiae GDP-mannose pyrophosphorylase. We examined the enzyme activity of a glutathione S-transferase fusion of each VIG9 gene to synthesize GDP mann ose in the cell extracts of a heterologous Escherichia coli expression syst em. We also developed a method for detecting the enzyme activity using a no n-radioactive substrate that would be applicable to high throughput screeni ng. (C) 2000 Elsevier Science B.V. All rights reserved.