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Journal of Biochemistry Advance Access originally published online on September 17, 2007
Journal of Biochemistry 2007 142(4):491-500; doi:10.1093/jb/mvm174
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© 2007 The Japanese Biochemical Society.

Function-unknown Glycoside Hydrolase Family 31 Proteins, mRNAs of which were Expressed in Rice Ripening and Germinating Stages, are {alpha}-Glucosidase and {alpha}-Xylosidase*

Hiroyuki Nakai1, Shigeki Tanizawa1, Tatsuya Ito1, Koutarou Kamiya1, Young-Min Kim1, Takeshi Yamamoto1, Kazuki Matsubara1, Makoto Sakai2, Hiroyuki Sato3, Tokio Imbe4, Masayuki Okuyama1, Haruhide Mori1, Yoshio Sano1, Seiya Chiba1 and Atsuo Kimura1,{dagger}

1Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589; 2National Agricultural Research Center for Kyushu Okinawa Region, Chikugo 833-0041; 3National Institute of Crop Science, Tsukuba 305-8518; and 4Tropical Agriculture Research Front, Japan International Center for Agricultural Science, Ishigaki, Okinawa 907-0002, Japan.

{dagger}To whom correspondence should be addressed. Tel: +81-11-706-2816, Fax: +81-11-706-2808, E-mail: kimura{at}abs.agr.hokudai.ac.jp

Received July 2, 2007; Accepted July 18, 2007


  Abstract

In rice (Oryza sativa L., var Nipponbare) seeds, there were three mRNAs encoding for function-unknown hydrolase family 31 homologous proteins (ONGX-H1, ONGX-H3 and ONGX-H4): ONGX-H1 mRNA was expressed in ripening stage and mRNAs of ONGX-H3 and ONGX-H4 were found in both the ripening and germinating stages [Nakai et al., (2007) Biochimie 89, 49–62]. This article describes that the recombinant proteins of ONGX-H1 (rONGXG-H1), ONGX-H3 (rONGXG-H3) and ONG-H4 (rONGXG-H4) were overproduced in Pichia pastoris as fusion protein with the {alpha}-factor signal peptide of Saccharomyces cerevisiae. Purified rONGXG-H1 and rONGXG-H3 efficiently hydrolysed malto-oligosaccharides, kojibiose, nigerose and soluble starch, indicating that ONGX-H1 and ONGX-H3 are {alpha}-glucosidases. Their substrate specificities were similar to that of ONG2, a main {alpha}-glucosidase in the dry and germinating seeds. The rONGXG-H1 and rONGX-H3 demonstrated the lower ability to adsorb to and degradation of starch granules than ONG2 did, suggesting that three {alpha}-glucosidases, different in action to starch granules, were expressed in ripening stage. Additionally, purified rONGXG-H4 showed the high activity towards {alpha}-xylosides, in particular, xyloglucan oligosaccharides. The enzyme hardly hydrolysed {alpha}-glucosidic linkage, so that ONGX-H4 was an {alpha}-xylosidase. {alpha}-Xylosidase encoded in rice genome was found for the first time.

Key Words: {alpha}-glucosidase, {alpha}-xylosidase, glycoside hydrolase family 31, Pichia pastoris, starch granule-binding

Abbreviations: GH, glycoside hydrolase family; 4-MUG, 4-methylumberyferyl {alpha}-d-glucopyranoside; ONG and ONX, Oryza sativa L., var Nipponbare {alpha}-glucosidase and {alpha}-xylosidase, respectively; ONGX-H, homologue of ONG or ONX; ong, onx and ongx-h, cDNA encoding ONG, ONX and ONGX-H, respectively; ORF, open reading frame; rONG, rONGX-H and rONX, recombinant proteins of ONG; ONGX-H and ONX, respectively; PPC, Pichia pastoris cells harbouring expression plasmid for rONGX-H production; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; PG, phenyl {alpha}-D-glucoside; pNPG, p-nitrophenyl {alpha}-D-glucopyranoside; pNPX, p-nitrophenyl {alpha}-D-xylopyranoside; XGOS, xyloglucan oligosaccharide


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