ABSTRACT
Openreading frame mj0608 of the Methanococcus jannaschii genome, recognized by its sequence similarity to that of the gene coding for class C inorganic pyrophosphatase in Bacillus subtilis, was cloned and over-expressed in Escherichia coli. The protein was purified and characterized by SDS-PAGE, M(r), and N-terminal sequence. Under suitable conditions it catalyzed the specific hydrolysis of PPi at about 600 micromol x min(-1) x mg(-1) at 25 degrees C, and at 8000 micromol x min(-1) x mg(-1) at 85 degrees C. Therefore this protein is a specific inorganic pyrophosphatase. The activities of Mg(2+), Mn(2+), Co(2+), and Zn(2+) ions as cofactors for hydrolysis of PPi were compared at pH 7.5 and 9.0. Unlike the class C pyrophosphatase of B. subtilis, this enzyme required no prior activation by low concentrations of Mn(2+) or Co(2+) ions. However, prior exposure to these ions afforded striking protection against inhibition by sodium fluoride, to which the enzyme was otherwise very sensitive.
Subject(s)
Cations, Divalent/pharmacology , Enzyme Inhibitors/pharmacology , Methanococcus/enzymology , Pyrophosphatases/antagonists & inhibitors , Pyrophosphatases/genetics , Sodium Fluoride/pharmacology , Amino Acid Sequence , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Catalysis/drug effects , Chelating Agents/pharmacology , Diphosphates/metabolism , Edetic Acid/pharmacology , Enzyme Activation/drug effects , Hydrogen-Ion Concentration , Hydrolysis/drug effects , Inorganic Pyrophosphatase , Kinetics , Metals/pharmacology , Methanococcus/genetics , Open Reading Frames/genetics , Pyrophosphatases/isolation & purification , Pyrophosphatases/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology , Substrate Specificity , TemperatureABSTRACT
The gene encoding an acid extracellular protease (AXP) from Yarrowia lipolytica (Candida olea) 148 was cloned and the complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AXP consists of 353 amino acids with an M, of 37427. The gene also encodes a putative 17 amino acid hydrophobic prepeptide and a 27 amino acid propeptide containing no potential N-glycosylation sites. The mature extracellular enzyme is produced by cleavage between Phe and Ala. AXP is a member of the aspartyl family of proteases. AXP shows homology to proteases of several fungal genera and to human progastricin. The coding sequence is preceded by a potential regulatory region of 1982 bp. Transcription of both AXP and alkaline extracellular protease genes of Y. lipolytica 148 is regulated by the pH of culture.
