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Cite: NAR/gkaa742 2020



PUL General Information

Literature Information

PUL ID

PUL0361

PubMed

10972187, Extremophiles. 2000 Aug;4(4):189-200. doi: 10.1007/pl00010711.

Characterization method

enzyme activity assay

Genomic accession number

CP007013.1

Nucelotide position range

997862-1008239

Substrate

starch,maltodextrin

Loci

THEMA_05005-THEMA_05030

Species

Thermotoga maritima/2336

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

- THEMA_05005 AHD18270.1 0 - 1662 (-) CP007013.1:997862-999524 -
- THEMA_05010 AHD18271.1 1896 - 3075 (+) CP007013.1:999758-1000937 -
- THEMA_05015 AHD18272.1 3151 - 4885 (+) CP007013.1:1001013-1002747 -
- THEMA_05020 AHD18273.1 4884 - 7383 (+) CP007013.1:1002746-1005245 -
- THEMA_05025 AHD18274.1 7379 - 8801 (+) CP007013.1:1005241-1006663 -
- THEMA_05030 AHD18275.1 8935 - 10378 (+) CP007013.1:1006797-1008240 -

Cluster number

1

Gene name

Gene position

Gene type

Found by CGCFinder?

- 1 - 1662 (-) TC: gnl|TC-DB|Q05839|8.A.9.1.1 Yes
- 1897 - 3075 (+) TC: gnl|TC-DB|Q9S5Y1|3.A.1.1.22 Yes
- 3152 - 4885 (+) TC: gnl|TC-DB|Q9X0T0|3.A.1.1.22 Yes
- 4885 - 7383 (+) TC: gnl|TC-DB|Q9X2F5|3.A.1.1.22 Yes
- 7380 - 8801 (+) CAZyme: GH13_20|CBM34|GH13 Yes
- 8936 - 10378 (+) CAZyme: GH4 Yes

PUL ID

PUL0361

PubMed

10972187, Extremophiles. 2000 Aug;4(4):189-200. doi: 10.1007/pl00010711.

Title

Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent alpha-glucosidase.

Author

Raasch C, Streit W, Schanzer J, Bibel M, Gosslar U, Liebl W

Abstract

The gene for the alpha-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hydrolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritima alpha-glucosidase has the unusual property of requiring NAD+ and Mn2+ for activity. Co2+ and Ni2+ also activated AglA, albeit less efficiently than Mn2+. T. maritima AglA represents the first example of a maltodextrin-degrading alpha-glucosidase with NAD+ and Mn2+ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or beta-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD+ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of D-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90 degrees C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50 degrees C and 70 degrees C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h.