Structure, expression and function of Allomyces arbuscula CDP II (metacaspase) gene.
Allomyces arbuscula, a primitive chytridiomycete fungus, has two Ca(2+)-dependent cysteine proteases, the CDP I and CDP II. We have cloned and analyzed the nucleotide sequence of CDP II gene and domain structure of the protein. Blast analysis of the sequence has shown that the protein belongs to a newly described member of caspase superfamily protein, the metacaspase, a CD clan of C14 family cysteine protease, we hence-forth name it as AMca 2 (Allomyces metacaspase 2). Southern hybridization studies have shown that the gene exists in a single copy per genome. The transcriptional analysis by Northern hybridization has confirmed our previous results that the protein is developmentally regulated, i.e. present in active growth phase but disappears during nutritional stress which also induces reproductive differentiation, indicating that the protein promotes cell growth, not death. The recombinant gene product expressed in Escherichiacoli has all the catalytic properties of native enzyme, i.e. sensitivity to protease inhibitors and substrate specificity. There is an absolute requirement of Ca(2+) for the activation of catalytic activity and the presence of R residue at the cleavage site (P1 position) in the substrate. The presence of a second basic residue, either R or K, in the P2 position strongly inhibits the catalytic activity which is stimulated by the presence of P and to a lesser extent G at this site. Peptide substrates with D at the cleavage site are not recognised and therefore not cleaved. The enzyme activity is inhibited by EDTA-EGTA, cysteine protease inhibitors and a specific peptide inhibitor Ac GVRCHCL TFA, but not by E64, although a potent inhibitor of cysteine proteases.
Ojha
Mukti
M
Department of Biochemistry, University of Geneva, Sciences II, 30 quai Ernest-Ansermet, 1211 Geneva 4, Switzerland. Mukti.Ojha@unige.ch
Cattaneo
Arlette
A
Hugh
Séverine
S
Pawlowski
Jan
J
Cox
Jos A
JA
D055134:Allomyces / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D018628:Gene Dosage; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003546:Cysteine Endopeptidases