Construction of chimeric cyclodextrin glucanotransferases from Bacillus circulans A11 and Paenibacillus macerans IAM1243 and analysis of their product specificity.
Three DNA fragments of 7919 base pairs containing genes for beta-cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), an iron III dicitrate transport protein-like protein and a partial coding sequence for putative ferrichrome ABC transporter from Bacillus circulans A11 were cloned and sequenced (GenBank Accession AF302787). The DNA sequence contained a CGTase open reading frame of 2139 base pairs, which encoded a polypeptide of 713 amino acid residues. The signal peptide constituted the N-terminal 27 amino acid residues. The amino acid sequence was highly homologous to that of Bacillus sp. 1011 with 98.7% identity. The cloned CGTase gene contained its own promoter that directed the expression of the gene in Escherichia coli host cells. Chimeric construction against the alpha-CGTase from B. macerans IAM1243 was carried out by means of three created restriction sites, XhoI, SpeI, and MfeI, introduced by mutagenesis in between domains A/B and C, C and D, and D and E, respectively, and the NdeI site within the domains A/B. The various chimeras with different combinations of domains and part of domains A/B were analyzed for their dextrinizing and CD-forming activities. Their activities were of three groups: chimeras with no dextrinizing and cyclization activities, chimeras with only dextrinizing activity, and chimeras with both dextrinizing and cyclization activities. Two chimeras in the latter group showed altered product specificity. The results located the amino acid segment essential for the product specificity at the C-terminal half of domains A/B. Further, the function of domains C and D in positioning domain E in the correct orientation and proximity to domains A/B is implicated.
Rimphanitchayakit
Vichien
V
Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. kpvr2@yahoo.com
Tonozuka
Takashi
T
Sakano
Yoshiyuki
Y
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase