Single Amino Acid Mutations Interchange the Reaction Specificities of Cyclodextrin Glycosyltransferase and the Acarbose-Modifying Enzyme Acarviosyl Transferase

Abstract

Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase Leemhuis, H.; Wehmeier, U.F.; Dijkhuizen, Lubbert Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. D-42079 Wuppertal, Germany ReceiVed May 15, 2004; ReVised Manuscript ReceiVed July 22, 2004 ABSTRACT: 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 R-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 µmol min -1 mg -1 ); acarbose is a strong inhibitor of CGTase and of most other R-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of R-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the R-amylase family, changed ATase into an enzyme with 4-R-glucanotransferase activity (3.4 µmol 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 µmol min -1 mg -1 ).The R-amylase family, or glycoside hydrolase family 13 (1), is a large family of enzymes acting on R-glycosidic bonds in starch and related compounds (2). About 20 different reaction specificities have been identified in this family, including hydrolysis of R-(1,4)-and R-(1,6)-glycosidic linkages (e.g., R-amylase and isoamylase, respectively), as well as the formation of R-(1,4)-and R-(1,6)-glycosidic bonds (e.g., amylosucrase, acarviosyl transferase, cyclodextrin glycosyltransferase, and branching enzyme, respectively; Figure 1) (2, 3). All members use an R-retaining double displacement mechanism (4) in which reactions proceed via a covalent glycosyl-enzyme intermediate (5). Glycosidic bond cleavage occurs between subsites -1 and +1 ( Figure 2A), and after cleavage the glycosyl reaction intermediate re...

Analysis of the key active subsites of glycoside hydrolase 13 family members

“…1,20 Replacement of H103 of Thermoanaerobacterium thermosulfurigenes CGTase by alanine leads to acaviosyl transferase activity, which suggests its importance in the reaction specificity. 21 2.2. The types of amino acid residues or the functional groups that constitute the À2, À1, +1, and +2 subsites of GH13 family members are not conserved…”

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

“…For CGTase, amylosucrase, neopullulanase, acarviosyltransferase, and the branching enzyme of family GH13 (3,13,96,101,102,104,105,206) and GS enzymes of family GH70, these regions have been proven to be important for product formation, giving further insights into the structural relatedness of GS and family GH13 enzymes (see below).…”

Evolution toward Small Molecule Inhibitor Resistance Affects Native Enzyme Function and Stability, Generating Acarbose-insensitive Cyclodextrin Glucanotransferase Variants

“…Substitution of this residue by glutamine may alter or disrupt this specific enzyme-inhibitor interaction, leading to reduced inhibition by acarbose. Interestingly, this residue is replaced in some GH13 acarbose-resistant glucanotransferases, suggesting an evolutionary role for acquired resistance (supplemental Table S1) (28,41).…”

“…Surprisingly, no His-140 mutant was identified from the screening as this histidine residue has been shown to be important for the strong inhibition by acarbose in Bacillus sp. 1011 and Thermoanaerobacterium thermosulfurigenes EM1 CGTases (27,28). His-140 was therefore targeted by saturation mutagenesis.…”