Two trehalose-hydrolyzing enzymes from Crenarchaeon Sulfolobus acidocaldarius exhibit distinct activities and affinities toward trehalose
Two archaeal trehalase-like genes, Saci1250 and Saci1816, belonging to glycoside hydrolase family 15 (GH15) from the acidophilic Crenarchaeon Sulfolobus acidocaldarius were expressed in Escherichia coli. The gene products showed trehalose-hydrolyzing activities, and the names SaTreH1 and SaTreH2 were assigned to Saci1816 and Saci1250 gene products, respectively. These newly identified enzymes functioned within a narrow range of acidic pH values at elevated temperatures, which is similar to the behavior of Euryarchaeota Thermoplasma trehalases. SaTreH1 displayed high KM and kcat values, whereas SaTreH2 had lower KM and kcat values despite a high degree of identity in their primary structures. A mutation analysis indicated that two glutamic acid residues in SaTreH1, E374 and E574, may be involved in trehalase catalysis because SaTreH1 E374Q and E574Q showed greatly reduced trehalose-hydrolyzing activities. Additional mutations substituting G573 and H575 residues with serine and glutamic acid residues, respectively, to mimic the TVN1315 sequence resulted in a decrease in trehalase activity and thermal stability. Taken together, the results indicated that Crenarchaea trehalases adopt active site structures that are similar to Euryarchaeota enzymes but have distinct molecular features. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 trehalases as well as other family enzymes and will provide insights into archaeal trehalose metabolism.
KeywordsArchaeal GH15 trehalase Expression Characterization Crenarchaea Sulfolobus acidocaldarius
We are grateful to Yukari Saisaka (High-Tech Research Center, Meiji Pharmaceutical University) for performing the N-terminal sequence analysis and Misa Ohno, Kazuaki Okawa, and Satoshi Wakita for their valuable suggestions.
This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Number 17 K07729); the Strategic Research Foundation Grant-aided Project for Private Universities of the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT) (Grant Number S1411005); the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan; and the Project Research Grant from the Research Institute of Science and Technology, Kogakuin University.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals.
- DeBosch BJ, Heitmeier MR, Mayer AL, Higgins CB, Crowley JR, Kraft TE, Chi M, Newberry EP, Chen Z, Finck BN, Davidson NO, Yarasheski KE, Hruz PW, Moley KH (2016) Trehalose inhibits solute carrier 2A (SLC2A) proteins to induce autophagy and prevent hepatic steatosis. Sci Signal 9:ra21. https://doi.org/10.1126/scisignal.aac5472 CrossRefPubMedPubMedCentralGoogle Scholar
- Jules M, Beltran G, François J, Parrou JL (2008) New insights into trehalose metabolism by Saccharomyces cerevisiae: NTH2 encodes a functional cytosolic trehalase, and deletion of TPS1 reveals Ath1p-dependent trehalose mobilization. Appl Environ Microbiol 74:605–614. https://doi.org/10.1128/AEM.00557-07 CrossRefPubMedGoogle Scholar
- Kopecka M, Kosek D, Kukacka Z, Rezabkova L, Man P, Novak P, Obsil T, Obsilova V (2014) Role of the EF-hand-like motif in the 14-3-3 protein-mediated activation of yeast neutral trehalase Nth1. J Biol Chem 289:13948–13961. https://doi.org/10.1074/jbc.M113.544551 CrossRefPubMedPubMedCentralGoogle Scholar
- Okawa K, Ohno M, Kashimura A, Kimura M, Kobayashi Y, Sakaguchi M, Sugahara Y, Kamaya M, Kino Y, Bauer PO, Oyama F (2016) Loss and gain of human acidic mammalian chitinase activity by nonsynonymous SNPs. Mol Biol Evol 33:3183–3193. https://doi.org/10.1093/molbev/msw198 CrossRefPubMedPubMedCentralGoogle Scholar
- Sakaguchi M, Matsushima Y, Nankumo T, Seino J, Miyakawa S, Honda S, Sugahara Y, Oyama F, Kawakita M (2014) Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification. AMB Express 4:5. https://doi.org/10.1186/2191-0855-4-5 CrossRefPubMedPubMedCentralGoogle Scholar
- Sakaguchi M, Shimodaira S, Ishida S, Amemiya M, Honda S, Sugahara Y, Oyama F, Kawakita M (2015) Identification of GH15 family thermophilic archaeal trehalases that function within a narrow acidic pH range. Appl Environ Microbiol 81:4920–4931. https://doi.org/10.1128/AEM.00956-15 CrossRefPubMedPubMedCentralGoogle Scholar
- Sakaguchi M, Matsushima Y, Nagamine Y, Matsuhashi T, Honda S, Okuda S, Ohno M, Sugahara Y, Shin Y, Oyama F, Kawakita M (2017) Functional dissection of the N-terminal sequence of Clostridium sp. G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure. Appl Microbiol Biotechnol 101:2415–2425. https://doi.org/10.1007/s00253-016-8024-4 CrossRefPubMedGoogle Scholar