PUL ID

PUL0176

PubMed

27302067, J Biol Chem. 2016 Aug 5;291(32):16438-47. doi: 10.1074/jbc.M116.727305. Epub 2016 Jun 14.

Characterization method

RT-PCR, enzyme activity assay, enzymatic product analysis

Genomic accession number

CP001736.1

Nucelotide position range

1994155-2001711

Substrate

isomaltose

Loci

Kfla_1895-Kfla_1900

Species

Kribbella flavida/182640

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

- Kfla_1895 ADB30989.1 0 - 2172 (-) CP001736.1:1994155-1996327 -
- Kfla_1896 ADB30990.1 2171 - 3329 (-) CP001736.1:1996326-1997484 -
- Kfla_1897 ADB30991.1 3337 - 4588 (-) CP001736.1:1997492-1998743 -
- Kfla_1898 ADB30992.1 4611 - 5436 (-) CP001736.1:1998766-1999591 -
- Kfla_1899 ADB30993.1 5428 - 6352 (-) CP001736.1:1999583-2000507 -
- Kfla_1900 ADB30994.1 6348 - 7557 (-) CP001736.1:2000503-2001712 -

Cluster number

1

Gene name

Gene position

Gene type

Found by CGCFinder?

- 1 - 2172 (-) CAZyme: GH31 Yes
- 2172 - 3329 (-) CAZyme: GH15 Yes
- 3338 - 4588 (-) TC: gnl|TC-DB|O07009|3.A.1.1.2 Yes
- 4612 - 5436 (-) TC: gnl|TC-DB|Q93J92|3.A.1.1.36 Yes
- 5429 - 6352 (-) TC: gnl|TC-DB|Q7WWQ9|3.A.1.1.20 Yes
- 6349 - 7557 (-) TF: DBD-Pfam|MarR No

PUL ID

PUL0176

PubMed

27302067, J Biol Chem. 2016 Aug 5;291(32):16438-47. doi: 10.1074/jbc.M116.727305. Epub 2016 Jun 14.

Title

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

Author

Tagami T, Miyano E, Sadahiro J, Okuyama M, Iwasaki T, Kimura A

Abstract

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