Conversion of the maltogenic alpha-amylase Novamyl into a CGTase.
Novamyl is a thermostable five-domain maltogenic alpha-amylase that shows sequence and structural homology with the cyclodextrin glycosyltransferases (CGTases). Comparing X-ray crystal structures of Novamyl and CGTases, two major differences in the active site cleft were observed: Novamyl contains a loop insertion consisting of five residues (residues 191-195) and the location of an aromatic residue known to be essential to obtain an efficient cyclization reaction. To convert Novamyl into a cyclodextrin (CD)-producing enzyme, the loop was deleted and two substitutions, F188L and T189Y, were introduced. Unlike the parent Novamyl, the obtained variant is able to produce beta-CD and showed an overall conversion of starch to CD of 9%, compared with CGTases which are able to convert up to 40%. The lower conversion compared with the CGTase is probably due to additional differences in the active site cleft and in the starch-binding E domain. A variant with only the five-residue loop deleted was not able to form beta-CD.
Beier
L
L
Novo Nordisk A/S, Novo Allé, DK-2800 Bagsvaerd, Denmark.
Svendsen
A
A
Andersen
C
C
Frandsen
T P
TP
Borchert
T V
TV
Cherry
J R
JR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003505:Cyclodextrins / Q000096:biosynthesis; D005964:Glucosyltransferases / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000378:metabolism; D047392:beta-Cyclodextrins
D001426:Bacterial Proteins; D003505:Cyclodextrins; D047392:beta-Cyclodextrins; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D006026:Glycoside Hydrolases; C120909:glucan 1,4-alpha-maltohydrolase; C031215:betadex