Structure–function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

The residue 179 is involved in product specificity of the Bacillus circulans DF 9R cyclodextrin glycosyltransferase

“…Moreover, the absence of side chains at positions 179 and 180 has been proposed as a strong requirement for substrate binding and cyclization reaction (Leemhuis et al 2002). However, we reported the presence of Gln instead of Gly at position 179 of the B. circulans DF 9R α/β-CGTase; this enzyme is the first CGTase displaying a natural mutant at this otherwise conserved position (Costa et al 2009). …”

“…The plasmid obtained was named pCR2.1/CGTase. Escherichia coli DH5α transformants were selected on lysogeny broth (LB) agar plates with kanamycin and X-Gal as described previously (Costa et al 2009). …”

“…CGTase gene amplification DNA was extracted and purified from B. circulans DF 9R (Costa et al 2009) and used as the template (5 ng for each polymerase chain reaction (PCR) reaction). The fragment encoding the CGTase without its signal peptide was amplified using 0.3 mM Nt-NcoI and Ct-XhoI primers (Table 1), 0.2 mM dNTPs, and 2.5 U platinum Pfx DNA polymerase in a 50-μl final volume.…”

“…The diversity of CDs obtained could be the consequence of specific amino acid substitutions, insertions, or deletions in the −3 and −7 subsites (Goh et al 2009;Kelly et al 2009;Li et al 2009a, b;Parsiegla et al 1998;Takada et al 2003;van der Veen et al 2000a, b). The −6 subsite (Y167, G179, G180, N193, and D196) is highly conserved in all CGTases whose primary structure is already known except that from B. circulans DF 9R (Costa et al 2009). Moreover, the absence of side chains at positions 179 and 180 has been proposed as a strong requirement for substrate binding and cyclization reaction (Leemhuis et al 2002).…”

Polysaccharide-synthesizing Glycosyltransferases and Carbohydrate Binding Modules: the case of Starch Synthase III

“…This family include different GTs from Archea, such as ancyclomaltodextrin glycosyltransferase [29]; several -cyclodextrin glycosyltransferases from bacteria, which cyclizes part of the -1,4 glucan chain [30,31] and two glucanotransferases, essential for maltose metabolism in plants and probably, playing a role in freezing tolerance [32]. CBM48 family is represented by GT from bacteria and eucaryota.…”