Abstract
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.
Highlights
Cyclobis-(136)-␣-nigerosyl (CNN),3 known as cycloalternan or cyclic tetrasaccharide, is a cyclic glucotetraose with alternate ␣-(136)- and ␣-(133)-glucosidic linkages (Fig. 1)
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
We revealed that the actinobacterium Kribbella flavida NBRC 14399T extracellularly produces CNN from starch, and intracellularly degrades CNN into glucose via isomaltose
Summary
Production and Degradation of CNN by K. flavida—K. flavida was cultured using soluble starch as the sole carbon source. The membrane fraction did not degrade CNN, the cell-free lysate produced isomaltose, glucose, and an oligosaccharide that may be a linearized CNN. This result indicates that K. flavida intracellularly catabolizes CNN via isomaltose. The site-directed mutations of Asp451 and Asp516 of Kfla1895 to Ala resulted in complete loss of the CNN hydrolyzing activity (Fig. 5B) These results implied that Asp451 and Asp516 of Kfla1895 were, respectively, catalytic nucleophile and acid/base as found for other GH31 enzymes. Substrate specificity analysis revealed that Kfla1896 displays the highest reaction rate with isomaltose, lower rates with panose and isomaltotriose, but almost no activity with other ␣-glucobioses (trehalose, kojibiose, nigerose, and maltose), CNN, or polysaccharides (dextran and soluble starch) (Table 1). Kfla1896 displayed a Km value for isomaltose that was similar to those of its other substrates, but a 10-fold higher kcat value for isomaltose than those
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