Function-unknown glycoside hydrolase family 31 proteins, mRNAs of which were expressed in rice ripening and germinating stages, are alpha-glucosidase and alpha-xylosidase.
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.
Nakai
Hiroyuki
H
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589; National Agricultural Research Center for Kyushu Okinawa Region, Chikugo 833-0041, Japan.
Tanizawa
Shigeki
S
Ito
Tatsuya
T
Kamiya
Koutarou
K
Kim
Young-Min
YM
Yamamoto
Takeshi
T
Matsubara
Kazuki
K
Sakai
Makoto
M
Sato
Hiroyuki
H
Imbe
Tokio
T
Okuyama
Masayuki
M
Mori
Haruhide
H
Sano
Yoshio
Y
Chiba
Seiya
S
Kimura
Atsuo
A
D003001:Cloning, Molecular; D018525:Germination / Q000502:physiology*; D012275:Oryza / Q000201:enzymology* / Q000254:growth & development / Q000502:physiology; D010843:Pichia; D012333:RNA, Messenger / Q000096:biosynthesis*; D012639:Seeds / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D014995:Xylosidases; C047831:alpha-D-xylosidase; D000520:alpha-Glucosidases