Function analysis of conserved amino acid residues in a Mn(2+)-dependent protein phosphatase, Pph3, from Myxococcus xanthus.
The Myxococcus xanthus protein phosphatase Pph3 belongs to the Mg(2+)- or Mn(2+)-dependent protein phosphatase (PPM) family. Bacterial PPMs contain three divalent metal ions and a flap subdomain. Putative metal- or phosphate-ion binding site-specific mutations drastically reduced enzymatic activity. Pph3 contains a cyclic nucleotide monophosphate (cNMP)-binding domain in the C-terminal region, and it requires 2-mercaptoethanol for phosphatase activity; however, the C-terminal deletion mutant showed high activity in the absence of 2-mercaptoethanol. The phosphatase activity of the wild-type enzyme was higher in the presence of cAMP than in the absence of cAMP, whereas a triple mutant of the cNMP-binding domain showed slightly lower activities than those of wild-type, without addition of cAMP. In addition, mutational disruption of a disulphide bond in the wild-type enzyme increased the phosphatase activity in the absence of 2-mercaptoethanol, but not in the C-terminal deletion mutant. These results suggested that the presence of the C-terminal region may lead to the formation of the disulphide bond in the catalytic domain, and that disulphide bond cleavage of Pph3 by 2-mercaptoethanol may occur more easily with cAMP bound than with no cAMP bound.
Department of Applied Biological Science, Kagawa University, Miki-cho, Kagawa, Japan.
Yumi
Y
Mori
Kaoru
K
Takegawa
Yoshio
Y
Kimura
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence / Q000235:genetics*; D000242:Cyclic AMP / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D016941:Myxococcus xanthus / Q000187:drug effects / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects
D000596:Amino Acids; D001426:Bacterial Proteins; D004220:Disulfides; D050505:Mutant Proteins; D008345:Manganese; D000242:Cyclic AMP; D010749:Phosphoprotein Phosphatases