title: Membrane-bound acid pyrophosphatase from Sulfolobus tokodaii, a thermoacidophilic archaeon: heterologous expression of the gene and characterization of the product.
abstract: 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.
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Fumitoshi
authors.initials: F
authors.lastname: Manabe
authors.forename: Yuko H
authors.initials: YH
authors.lastname: Itoh
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Shoun
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Wakagi
mesh_terms: 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*
chemical_list: D019843:Archaeal Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D001414:Bacitracin; D011755:Pyrophosphatases; C023082:dolichyldiphosphatase
references.citation: Proc Natl Acad Sci U S A. 1971 Dec;68(12):3223-7
references.pmid: 4332017
references.citation: Biochim Biophys Acta. 1988 Jun 9;947(2):307-33
references.pmid: 3285892
references.citation: Biochim Biophys Acta. 1995 Jun 1;1230(1-2):31-7
references.pmid: 7612641
references.citation: J Bacteriol. 1989 Dec;171(12):6710-9
references.pmid: 2512283
references.citation: J Biochem. 1993 Sep;114(3):329-33
references.pmid: 8282721
references.citation: Arch Mikrobiol. 1972;84(1):54-68
references.pmid: 4559703
references.citation: Eur J Biochem. 1992 Jul 15;207(2):741-6
references.pmid: 1321720
references.citation: Biosci Biotechnol Biochem. 1998 Dec;62(12):2408-14
references.pmid: 9972267
references.citation: Lipids. 1995 Apr;30(4):339-44
references.pmid: 7609602
references.citation: Curr Microbiol. 2000 Jan;40(1):57-60
references.pmid: 10568805
references.citation: J Mol Biol. 1996 Jul 19;260(3):289-98
references.pmid: 8757792
references.citation: Glycobiology. 1997 Oct;7(7):897-904
references.pmid: 9363431
references.citation: Biochim Biophys Acta. 1985 Jul 11;817(1):33-41
references.pmid: 3159431
references.citation: J Biol Chem. 2005 Aug 12;280(32):28852-7
references.pmid: 15946938
references.citation: J Biol Chem. 1973 Jun 10;248(11):3940-5
references.pmid: 4350651
references.citation: J Biol Chem. 2008 Jun 13;283(24):16573-83
references.pmid: 18411271
references.citation: J Bioenerg Biomembr. 2004 Feb;36(1):143-50
references.pmid: 15168618
references.citation: J Bacteriol. 1989 Nov;171(11):6106-16
references.pmid: 2478523
references.citation: Biochim Biophys Acta. 1992 Apr 17;1120(3):289-96
references.pmid: 1315573
references.citation: Anal Biochem. 1975 Feb;63(2):607-13
references.pmid: 1122033
references.citation: J Biol Chem. 2002 Nov 8;277(45):42530-9
references.pmid: 12186869
references.citation: Microbiol Mol Biol Rev. 2005 Sep;69(3):393-425
references.pmid: 16148304