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Cite: NAR/gkaa742 2020



PUL General Information

Literature Information

PUL ID

PUL0423

PubMed

10960102, J Bacteriol. 2000 Sep;182(18):5172-9. doi: 10.1128/JB.182.18.5172-5179.2000.

Characterization method

clone and expression,enzyme activity assay

Genomic accession number

AF039487

Nucelotide position range

471-4846

Substrate

cellobiose

Loci

cbpA-gghA-amyB

Species

Thermotoga neapolitana/2337

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

cbpA - AAB95491.2 471 - 2910 (+) AF039487.2:942-3381 2.4.1.20
gghA - AAB95492.2 3012 - 4347 (+) AF039487.2:3483-4818 3.2.1.21
amyB - AAF37330.1 4570 - 4846 (+) AF039487.2:5041-5317 -

Cluster number

1

Gene name

Gene position

Gene type

Found by CGCFinder?

- 472 - 2910 (+) CAZyme: GH94 Yes
- 3013 - 4347 (+) TC: gnl|TC-DB|Q86Z14|8.A.49.1.2 Yes
- 4571 - 4846 (+) CDS No

PUL ID

PUL0423

PubMed

10960102, J Bacteriol. 2000 Sep;182(18):5172-9. doi: 10.1128/JB.182.18.5172-5179.2000.

Title

Cloning and characterization of the glucooligosaccharide catabolic pathway beta-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana.

Author

Yernool DA, McCarthy JK, Eveleigh DE, Bok JD

Abstract

Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghA's K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.