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Molecular cloning and biochemical characterization of a 3′(2′),5′‐bisphosphate nucleotidase from Debaryomyces hansenii
Author(s) -
Aggarwal Monika,
Bansal Parmil K.,
Mondal Alok K.
Publication year - 2005
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1223
Subject(s) - debaryomyces hansenii , biology , biochemistry , yeast , molecular cloning , saccharomyces cerevisiae , enzyme , microbiology and biotechnology , open reading frame , gene , peptide sequence
The enzyme 3′(2′),5′‐bisphosphate nucleotidase catalyses a reaction that converts 3′‐phosphoadenosine‐5′‐phosphate (PAP) to adenosine‐5′‐phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae , the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2 , from the halotolerant yeast Debaryomyces hansenii . DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP ( K m 17 µ M ) and PAPS ( K m 48 µ M ) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions. The nucleotide sequence of DHAL2 gene has been submitted to Genbank (Accession No. AY340817). Copyright © 2005 John Wiley & Sons, Ltd.