Two Novel Glycoside Hydrolases Responsible for the Catabolism of Cyclobis-(1→6)-α-nigerosyl

J Biol Chem. 2016 Aug 5;291(32):16438-47. doi: 10.1074/jbc.M116.727305. Epub 2016 Jun 14.


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.

Keywords: actinobacteria; bacterial metabolism; carbohydrate metabolism; cyclic oligosaccharide; enzyme catalysis; enzyme kinetics; gene expression; glycoside hydrolase; glycosyltransferase; starch.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinobacteria* / enzymology
  • Actinobacteria* / genetics
  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / metabolism
  • Genome, Bacterial / physiology*
  • Glucans / genetics
  • Glucans / metabolism*
  • Glycoside Hydrolases* / genetics
  • Glycoside Hydrolases* / metabolism
  • Multigene Family / physiology*


  • Bacterial Proteins
  • Glucans
  • cyclic nigerosylnigerose
  • Glycoside Hydrolases