Question

Make an assignment on pullulanase related to conserved domains,
identical protein groups,
and protein clustere​

Answer 1

Answer:

A gene encoding a cyclomaltodextrinase (neopullulanase) was cloned from the thermoacidophilic bacterium Alicyclobacillus acidocaldariusATCC27009 and its nucleotide sequence was determined. The encoded CdaA protein lacked an N-terminal signal sequence and aligned well with a family of bacterial proteins described as maltogenic α-amylases, neopullulanases or cyclomaltodextrinases. Escherichia colicells harboring the cloned cdaA gene produced a 66-kDa protein that degraded pullulan in a sodium dodecyl sulfate-polyacrylamide gel. A. acidocaldarius cells grown on maltose, soluble starch or pullulan synthesized the same protein. Neopullulanase activity of the protein was cytoplasmic and its pH optimum of 5.5 was close to the pH value of the cytoplasm. CdaA degraded cyclomaltodextrins rapidly and pullulan (to panose) more slowly. It is proposed that CdaA functions as a cytoplasmic cyclomaltodextrinase (EC 3.2.1.54).

Family 13 of the glycosylhydrolases (also α-amylase family) is huge (reviewed in [1]). Among others, it contains α-amylases (EC 3.2.1.1), which hydrolyze internal α-1,4-glycosidic bonds in amylose and amylopectin, amylopullulanases (EC 3.2.1.1/41), which cleave with one type of active center both α-1,4- and α-1,6-glycosidic bonds in starch and α-1,6-glycosidic bonds in pullulan, neopullulanases (EC 3.2.1.135), which break α-1,4-glycosidic bonds in pullulan with the formation of the trisaccharide panose, and cyclomaltodextrinases (EC 3.2.1.54), which linearize cyclomaltodextrins by hydrolyzing α-1,4-glycosidic bonds. Within family 13, cyclomaltodextrinases and neopullulanases are closely related to eachother .

In the course of studies on the function of a cluster of genes involved in starch degradation by the thermoacidophile Alicyclobacillus acidocaldarius ATCC27009 [2–4], we identified a gene, cdaA, that translates into a protein belonging to a new sub-group of cyclomaltodextrinases-neopullulanases-maltogenic α-amylases from the α-amylase family. We report here that CdaA activity is cytoplasmic and propose that CdaA functions as a cytoplasmic cyclomaltodextrinase. Like most of its sub-family members, it degraded besides cyclomaltodextrins also pullulan slowly to panose. Most likely, 12 bacterial enzymes closely related to CdaA ([5–13] and Swiss protein accession numbers P32818, O82982 and Q59226) function also as cytoplasmic cyclomaltodextrinases-oligomaltodextrinases.