A Candida albicans cyclic nucleotide phosphodiesterase: cloning and expression in Saccharomyces cerevisiae and biochemical characterization of the recombinant enzyme

• Published in: Microbiology (Society for General Microbiology) 1994-07, Vol.140 (7), p.1533-1542
• Main Authors: HOYER, L. L, CIESLINSKI, L. B, MCLAUGHLIN, M. M, TORPHY, T. J, SHATZMAN, A. R, LIVI, G. P
• Format: Article
• Language: English
• Subjects: 3',5'-Cyclic-AMP Phosphodiesterases - genetics; 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Amino Acid Sequence; Base Sequence; Candida albicans - enzymology; Candida albicans - genetics; Cations, Divalent - metabolism; Cloning, Molecular; Cyclic AMP - metabolism; Cyclic GMP - metabolism; Cyclic Nucleotide Phosphodiesterases, Type 1; Genes, Fungal - genetics; Genetic Complementation Test; Molecular Sequence Data; Phosphoric Diester Hydrolases; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Species Specificity
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-140-7-1533

1 Department of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA 2 Department of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA Author for correspondence: L. L. Hoyer. Tel: [phone]. Fax: [phone]. e-mail: hoyer{at}genome.lbl.gov ABSTRACT We have cloned a Candida albicans gene, which encodes a cyclic nucleotide phosphodiesterase (PDEase), by complementation in a Saccharomyces cerevisiae PDEase-deficient mutant. The deduced amino acid sequence is similar to that of the low-affinity PDEase of S. cerevisiae (PDE1) and the cyclic nucleotide PDEase (PD) of Dictyostelium discoideum. Biochemical analysis of recombinant protein produced in S. cerevisiae indicated that the enzyme behaves as a PDE1 homologue: it hydrolyses both cAMP ( K m = 0.49 mM) and cGMP ( K m = 0.25 mM), does not require divalent cations for maximal activity and is only moderately inhibited by millimolar concentrations of standard PDEase inhibitors. Based on these data, we designate the C. albicans we have cloned, PDE1. Low-stringency genomic Southern blots showed cross-hybridization between C. albicans PDE1 and DNA from Candida stellatoidea , but not with DNA from S. cerevisiae or several closely related Candida species. Keywords: Candida albicans , cyclic nucleotide phosphodiesterase, morphogenesis The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number L12045 (Gb_pl:YSAPDE1X). Present address : Lawrence Berkeley Laboratory, Mail Stop 74-157, 1 Cyclotron Road, Berkeley, CA 94720, USA.