Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase.
Acarviosyl transferase (ATase) from Actinoplanes sp. SE50/110 is a bacterial enzyme that transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors. The enzyme exhibits 42% sequence identity with cyclodextrin glycosyltransferases (CGTase), and both enzymes are members of the alpha-amylase family, a large clan of enzymes acting on starch and related compounds. ATase is virtually inactive on starch, however. In contrast, ATase is the only known enzyme to efficiently use acarbose as substrate (2 micromol min(-1) mg(-1)); acarbose is a strong inhibitor of CGTase and of most other alpha-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of alpha-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the alpha-amylase family, changed ATase into an enzyme with 4-alpha-glucanotransferase activity (3.4 micromol min(-1) mg(-1)). Moreover, this mutation introduced cyclodextrin-forming activity into ATase, converting 2% of starch into cyclodextrins. The opposite experiment, removing this typical His side chain in CGTase (H140A), introduced acarviosyl transferase activity in CGTase (0.25 micromol min(-1) mg(-1)).
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Hans
H
Leemhuis
Udo F
UF
Wehmeier
Lubbert
L
Dijkhuizen
D020909:Acarbose / Q000378:metabolism*; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D002240:Carbohydrate Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005964:Glucosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics; D005998:Glycine / Q000235:genetics; D008848:Micromonosporaceae / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013912:Threonine / Q000235:genetics
D005973:Glutamine; D013912:Threonine; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C101444:acarbose 7-phosphotransferase; D020909:Acarbose; D005998:Glycine