Two Novel Glycoside Hydrolases Responsible for the Catabolism of Cyclobis-(1→6)-α-nigerosyl.
The actinobacterium Kribbella flavida NBRC 14399(T) produces cyclobis-(1→6)-α-nigerosyl (CNN), a cyclic glucotetraose with alternate α-(1→6)- and α-(1→3)-glucosidic linkages, from starch in the culture medium. We identified gene clusters associated with the production and intracellular catabolism of CNN in the K. flavida genome. One cluster encodes 6-α-glucosyltransferase and 3-α-isomaltosyltransferase, which are known to coproduce CNN from starch. The other cluster contains four genes annotated as a transcriptional regulator, sugar transporter, glycoside hydrolase family (GH) 31 protein (Kfla1895), and GH15 protein (Kfla1896). Kfla1895 hydrolyzed the α-(1→3)-glucosidic linkages of CNN and produced isomaltose via a possible linear tetrasaccharide. The initial rate of hydrolysis of CNN (11.6 s(-1)) was much higher than that of panose (0.242 s(-1)), and hydrolysis of isomaltotriose and nigerose was extremely low. Because Kfla1895 has a strong preference for the α-(1→3)-isomaltosyl moiety and effectively hydrolyzes the α-(1→3)-glucosidic linkage, it should be termed 1,3-α-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of β-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The kcat/Km for isomaltose (4.81 ± 0.18 s(-1) mm(-1)) was 6.9- and 19-fold higher than those for panose and isomaltotriose, respectively. These results indicate that Kfla1896 is a new GH15 enzyme with high substrate specificity for isomaltose, suggesting the enzyme should be designated an isomaltose glucohydrolase. This is the first report to identify a starch-utilization pathway that proceeds via CNN.
From the College of Agriculture, Food and Environment Sciences, Rakuno Gakuen University, Ebetsu 069-8501 and t-tagami@rakuno.ac.jp.
Takayoshi
T
Tagami
the Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Eri
E
Miyano
the Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Juri
J
Sadahiro
the Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Masayuki
M
Okuyama
From the College of Agriculture, Food and Environment Sciences, Rakuno Gakuen University, Ebetsu 069-8501 and.
Tomohito
T
Iwasaki
the Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan kimura@abs.agr.hokudai.ac.jp.
Atsuo
A
Kimura
D039903:Actinobacteria / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial / Q000502:physiology*; D005936:Glucans / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family / Q000502:physiology*
D001426:Bacterial Proteins; D005936:Glucans; C516208:cyclic nigerosylnigerose; D006026:Glycoside Hydrolases
actinobacteria; bacterial metabolism; carbohydrate metabolism; cyclic oligosaccharide; enzyme catalysis; enzyme kinetics; gene expression; glycoside hydrolase; glycosyltransferase; starch