title: Identification of GH15 Family Thermophilic Archaeal Trehalases That Function within a Narrow Acidic-pH Range.
abstract: Two glucoamylase-like genes, TVN1315 and Ta0286, from the archaea Thermoplasma volcanium and T. acidophilum, respectively, were expressed in Escherichia coli. The gene products, TVN1315 and Ta0286, were identified as archaeal trehalases. These trehalases belong to the CAZy database family GH15, although they have putative (α/α)6 barrel catalytic domain structures similar to those of GH37 and GH65 family trehalases from other organisms. These newly identified trehalases function within a narrow range of acidic pH values (pH 3.2 to 4.0) and at high temperatures (50 to 60°C), and these enzymes display Km values for trehalose higher than those observed for typical trehalases. These enzymes were inhibited by validamycin A; however, the inhibition constants (Ki) were higher than those of other trehalases. Three TVN1315 mutants, corresponding to E408Q, E571Q, and E408Q/E571Q mutations, showed reduced activity, suggesting that these two glutamic acid residues are involved in trehalase catalysis in a manner similar to that of glucoamylase. To date, TVN1315 and Ta0286 are the first archaeal trehalases to be identified, and this is the first report of the heterologous expression of GH15 family trehalases. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 family enzymes as well as glycoside hydrolase family enzymes; additionally, these enzymes provide insight into archaeal trehalose metabolism.
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan bt11532@ns.kogakuin.ac.jp.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Sakaguchi
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Shimodaira
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
authors.forename: Shin-Nosuke
authors.initials: SN
authors.lastname: Ishida
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
authors.forename: Miko
authors.initials: M
authors.lastname: Amemiya
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Honda
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
authors.forename: Yasusato
authors.initials: Y
authors.lastname: Sugahara
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Oyama
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Kawakita
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D016000:Cluster Analysis; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013696:Temperature; D013822:Thermoplasma / Q000201:enzymology* / Q000235:genetics; D014198:Trehalase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014199:Trehalose / Q000378:metabolism
chemical_list: D019641:DNA, Archaeal; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D011994:Recombinant Proteins; C030790:validamycin A; D007294:Inositol; D014199:Trehalose; D014198:Trehalase