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Journal of Biochemistry Advance Access published online on September 17, 2007
Journal of Biochemistry, 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,* *

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 Agriculitural Science, Ishigaki, Okinawa 907-0002

Corresponding author**: Atsuo Kimura: Mailing address: Graduate School of Agriculture, Hokkaido University,Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan, Telephone number: +81-11-706-2816, Fax number: +81-11-706-2808, E mail address: 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 of ripening and germinating stages [Nakai et al., (2007) Biochimie 89, 49-62]. This paper 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 hydrolyzed maltooligosaccharides, 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 degrade 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 toward {alpha}-xylosides, in particular, xyloglucan oligosaccharides. The enzyme hardly hydrolyzed {alpha}-glucosidic linkage, so that ONGX-H4 was an {alpha}-xylosidase. {alpha}-Xylosidase encoded in rice genome was found for first time.

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


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