Membrane-bound acid pyrophosphatase from Sulfolobus tokodaii, a thermoacidophilic archaeon: heterologous expression of the gene and characterization of the product.
Membranes of Sulfolobus tokodaii, a thermoacidophilic archaeon that grows optimally at pH 2-3, 75-80 degrees C, show the ability to hydrolyze PPi with an optimum pH of 2-3. This acid PPase is proposed to be a dolicholpyrophosphatase that participates in glycoprotein biosynthesis. In the present study, the archaeal membranes hydrolyzed isopentenylpyrophosphate and geranylpyrophosphate, compounds related to dolicholpyrophosphate, at pH 3. However, the dolicholpyrophosphate-binding antibiotic bacitracin failed to inhibit the acid PPase. To investigate further the function and structure of the acid PPase, the gene was cloned and heterologously expressed in Escherichia coli. The membranes from recombinant E. coli showed PPase activity with similar pH and temperature dependence, substrate specificity, and kinetic parameters to those reported for Sulfolobus membranes. The acid PPase was solubilized and purified to electrophoretic homogeneity from the recombinant E. coli. The purified enzyme showed similar K(m) values for PPi, ATP, and ADP to the membrane-bound enzyme. Lipids from the Sulfolobus membranes enhanced the activity to about threefold. Studies involving deletion mutants indicated that basic amino acids in the N-terminal (Arg2 and Lys3), as well as the residues (4th-69th) possibly twice-spanning the membrane, are essential for integration of the enzyme into membranes.
Manabe
Fumitoshi
F
Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Itoh
Yuko H
YH
Shoun
Hirofumi
H
Wakagi
Takayoshi
T
D020816:Amino Acid Motifs; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001414:Bacitracin / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli; D019766:Genes, Archaeal; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D011755:Pyrophosphatases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*
D019843:Archaeal Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D001414:Bacitracin; D011755:Pyrophosphatases; C023082:dolichyldiphosphatase
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