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

ABSTRACT: The actinobacterium Kribbella flavida NBRC 14399(T) produces cyclobis-(1?6)-?-nigerosyl (CNN), a cyclic glucotetraose with alternate ?-(1?6)- and ?-(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-?-glucosyltransferase and 3-?-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 ?-(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 ?-(1?3)-isomaltosyl moiety and effectively hydrolyzes the ?-(1?3)-glucosidic linkage, it should be termed 1,3-?-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of ?-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.

SUBMITTER: Tagami T 

PROVIDER: S-EPMC4974359 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature