Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii.
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 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.
Aggarwal
Monika
M
Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India.
Bansal
Parmil K
PK
Mondal
Alok K
AK
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D018021:Lithium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D009708:Nucleotidases / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000494:pharmacology
D004271:DNA, Fungal; D011994:Recombinant Proteins; D012965:Sodium Chloride; D000244:Adenosine Diphosphate; C010840:adenosine 3'-phosphate-5'-phosphate; D009708:Nucleotidases; C053354:bisphosphoadenylate 3'-nucleotidase; D018021:Lithium Chloride