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Cite: NAR/gkaa742 2020



PUL General Information

Literature Information

PUL ID

PUL0122

PubMed

9741105, Syst Appl Microbiol. 1998 Mar;21(1):1-11. doi: 10.1016/s0723-2020(98)80002-7.

Characterization method

enzyme activity assay

Genomic accession number

NC_023151.1

Nucelotide position range

1598265-1608135

Substrate

alpha-galactoside,beta-galactoside,raffinose,melibiose

Loci

THEMA_RS08275-THEMA_RS08300

Species

Thermotoga maritima/2336

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

- THEMA_RS08275 WP_004080133.1 0 - 1950 (+) NC_023151.1:1598265-1600215 -
- THEMA_RS08280 WP_004080134.1 1964 - 2945 (+) NC_023151.1:1600229-1601210 -
- THEMA_RS08285 WP_010865279.1 2941 - 6205 (+) NC_023151.1:1601206-1604470 -
- THEMA_RS08290 WP_004080136.1 6210 - 7869 (+) NC_023151.1:1604475-1606134 -
galT THEMA_RS08295 WP_004080138.1 7865 - 8822 (+) NC_023151.1:1606130-1607087 -
- THEMA_RS08300 WP_004080140.1 8818 - 9871 (+) NC_023151.1:1607083-1608136 2.7.1.6

Cluster number

1

Gene name

Gene position

Gene type

Found by CGCFinder?

- 1 - 1950 (+) CAZyme: GH42 Yes
- 1965 - 2945 (+) TC: gnl|TC-DB|Q9WXS9|3.A.1.5.13 Yes
- 2942 - 6205 (+) CAZyme: GH2 Yes
- 6211 - 7869 (+) CAZyme: GH36 Yes
galT 7866 - 8822 (+) CDS No
- 8819 - 9871 (+) CDS No

PUL ID

PUL0122

PubMed

9741105, Syst Appl Microbiol. 1998 Mar;21(1):1-11. doi: 10.1016/s0723-2020(98)80002-7.

Title

Properties of an alpha-galactosidase, and structure of its gene galA, within an alpha-and beta-galactoside utilization gene cluster of the hyperthermophilic bacterium Thermotoga maritima.

Author

Liebl W, Wagner B, Schellhase J

Abstract

Thermotoga maritima represents one of the few hyperthermophilic bacteria currently known. The chromosomal alpha-galactosidase gene of T. maritima strain MSB8 has been cloned and its nucleotide sequence was determined. The gene, designated galA, has coding capacity for a 552 residue polypeptide with a calculated molecular mass of 63,653 Da. GalA was found to be flanked by other genes probably involved in galactoside breakdown and utilization. The previously sequenced beta-galactosidase gene, lacZ, is localized immediately upstream of galA while two open reading frames that putatively encode enzymes of galactose catabolism, i.e. galactose-1-phosphate uridylytransferase (galT) and galactokinase (galK), were found downstream of galA. The identified genes are extremely close together or even overlap and have the same orientation, so they could all be part of one galactoside utilization operon of T. maritima MSB8. GalA displayed low-level amino acid sequence similarity with alpha-galactoside of glycosyl hydrolase family 36. However, GalA is smaller than the other members of this enzyme family. The galA gene was expressed in Escherichia coli and the recombinant alpha-galactosidase was purified and characterized. The molecular mass of the recombinant enzyme was estimated at about 62 kDa by denaturting gel electrophoresis. Maximal hydrolysis of the chromogenic substrate p-nitrophenyl-alpha-D-galactopyranoside was measured at pH 5.0-5.5 and 90-95 degrees C (5 min assay). Divalent cations were not required for activity. The enzyme released galactose from raffinose, melibiose and the synthetic substrates p-nitrophenyl-and omicron-nitrophenyl-alpha-D-galactopyranoside. The T. maritima alpha-galactosidase thus was highly specific for the galactose moiety and the alpha-anomeric configuration of the glycosidic linkage. Its extreme thermal stability (t 1/2 = 6.5 h at 85 degrees C) makes this enzyme an interesting candidate for biotechnological applications.