Abstract
Clostridium sp. G0005 glucoamylase (CGA) is composed of a β-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N-terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N-terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the N-terminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.
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References
Adam AC, Latorre-García L, Polaina J (2004) Structural analysis of glucoamylase encoded by the STA1 gene of Saccharomyces cerevisiae (var. diastaticus). Yeast 21:379–388. doi:10.1002/yea.1102
Aleshin AE, Feng PH, Honzatko RB, Reilly PJ (2003) Crystal structure and evolution of a prokaryotic glucoamylase. J Mol Biol 327:61–73. doi:10.1016/S0022-2836(03)00084-6
Bott R, Saldajeno M, Cuevas W, Ward D, Scheffers M, Aehle W, Karkehabadi S, Sandgren M, Hansson H (2008) Three-dimensional structure of an intact glycoside hydrolase family 15 glucoamylase from Hypocrea jecorina. Biochemistry 47:5746–5754. doi:10.1021/bi702413k
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 7:248–254
Carroll JD, Pastuszak I, Edavana VK, Pan YT, Elbein AD (2007) A novel trehalase from Mycobacterium smegmatis-purification, properties, requirements. FEBS J 274:1701–1714. doi:10.1111/j.1742-4658.2007.05715.x
Coutinho PM, Reilly PJ (1997) Glucoamylase structural, functional, and evolutionary relationships. Proteins 29:334–347. doi:10.1002/(SICI)1097-0134(199711)29:3<334::AID-PROT7>3.0.CO;2-A
Dalbøge H, Bayne S, Pedersen J (1990) In vivo processing of N-terminal methionine in E. coli. FEBS Lett 266:1–3. doi:10.1016/0014-5793(90)90001-B
Hirel PH, Schmitter MJ, Dessen P, Fayat G, Blanquet S (1989) Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc Natl Acad Sci U S A 86:8247–8251
Hiromi K, Nitta Y, Numata C, Ono S (1973) Subsite affinities of glucoamylase: examination of the validity of the subsite theory. Biochim Biophys Acta 302:362–375
Kumar P, Satyanarayana T (2009) Microbial glucoamylases: characteristics and applications. Crit Rev Biotechnol 29:225–255. doi:10.1080/07388550903136076
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0
Li Z, Wei P, Cheng H, He P, Wang Q, Jiang N (2014) Functional role of β domain in the Thermoanaerobacter tengcongensis glucoamylase. Appl Microbiol Biotechnol 98:2091–2099. doi:10.1007/s00253-013-5051-2
Lin SC, Liu WT, Liu SH, Chou WI, Hsiung BK, Lin IP, Sheu CC, Dah-Tsyr Chang M (2007) Role of the linker region in the expression of Rhizopus oryzae glucoamylase. BMC Biochem 8:9. doi:10.1186/1471-2091-8-9
Marín-Navarro J, Polaina J (2011) Glucoamylases: structural and biotechnological aspects. Appl Microbiol Biotechnol 89:1267–1273. doi:10.1007/s00253-010-3034-0
Ohnishi H, Matsumoto H, Sakai H, Ohta T (1994) Functional roles of Trp337 and Glu632 in Clostridium glucoamylase, as determined by chemical modification, mutagenesis, and the stopped-flow method. J Biol Chem 269:3503–3510
Sakaguchi M, Matsushima Y, Nankumo T, Seino J, Miyakawa S, Honda S, Sugahara Y, Oyama F, Kawakita M (2014a) Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification. AMB Express 4:5. doi:10.1186/2191-0855-4-5
Sakaguchi M, Osaku K, Maejima S, Ohno N, Sugahara Y, Oyama F, Kawakita M (2014b) Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1. AMB Express 4:59. doi:10.1186/s13568-014-0059-2
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. doi:10.1128/AEM.00956-15
Sambrook J, Russell DW (2012) Molecular cloning: a laboratory manual, 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sevcík J, Solovicová A, Hostinová E, Gasperík J, Wilson KS, Dauter Z (1998) Structure of glucoamylase from Saccharomycopsis fibuligera at 1.7 Å resolution. Acta Crystallogr D Biol Crystallogr 54:854–866. doi:10.1107/S0907444998002005
Svensson B, Larsen K, Gunnarsson A (1986) Characterization of a glucoamylase G2 from Aspergillus niger. Eur J Biochem 154:497–502. doi:10.1111/j.1432-1033.1986.tb09425.x
Wilkinson AJ, Fersht AR, Blow DM, Winter G (1983) Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation. Biochemistry 22:3581–3586
Acknowledgements
We are grateful to Yukari Saisaka (High-Tech Research Center, Meiji Pharmaceutical University) for performing the N-terminal sequence analysis and Syoma Sakamoto, Kazuaki Okawa, Satoshi Wakita, and Masahiro Kimura for their valuable suggestions and technical assistance.
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This study was supported in part by a grant from the Strategic Research Foundation Grant-aided Project for Private Universities of the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT) (S1411005); by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan; and by the Project Research Grant from the Research Institute of Science and Technology, Kogakuin University.
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Sakaguchi, M., Matsushima, Y., Nagamine, Y. et al. 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 (2017). https://doi.org/10.1007/s00253-016-8024-4
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DOI: https://doi.org/10.1007/s00253-016-8024-4