Engineering of factors determining α‐amylase and cyclodextrin glycosyltransferase specificity in the cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1

Abstract

The starch-degrading enzymes A-amylase and cyclodextrin glycosyltransferase (CGTase) are functionally and structurally closely related, with CGTases containing two additional domains (called D and E) compared to the three domains of A-amylases (A, B and C). Amino acid residue 196 (Thermoanaerobacterium thermosulfurigenes EM1 CGTase numbering) occupies a dominant position in the active-site cleft. All A-amylases studied have a small residue at this position (Gly, Leu, Ser, Thr or Val), in contrast to CGTases which have a more bulky aromatic residue (Tyr or Phe) at this position, which is highly conserved. Characterization of the F196G mutant CGTase of T. thermosulfurigenes EM1 revealed that, for unknown reasons, apart from the F196G mutation, domain E as well as a part of domain D had become deleted [mutant F196G(∆′DE)]. This, nevertheless, did not prevent the purification of a stable and active mutant CGTase protein (62 kDa). The mutant protein was more similar to an A-amylase protein in terms of the identity of residue 196, and in the domain structure containing, however, some additional C-terminal structure. The mutant showed a strongly reduced temperature optimum. Due to a frameshift mutation in mutant F196G, a separate protein of 19 kDa with the DE domains was also produced. Mutant F196G(∆′DE) displayed a strongly reduced raw-starch-binding capacity, similar to the situation in most A-amylases that lack a raw-starch-binding E domain. Compared to wild-type CGTase, cyclization, coupling and disproportionation activities had become drastically reduced in the mutant F196G(∆′DE), but its saccharifying activity had doubled, reaching the highest level ever reported for a CGTase. Under industrial production process conditions, wild-type CGTase converted starch into 35% cyclodextrins and 11% linear oligosaccharides (glucose, maltose and maltotriose), whereas mutant F196G(∆′DE) converted starch into 21% cyclodextrins and 18% into linear oligosaccharides. These biochemical characteristics indicate a clear shift from CGTase to A-amylase specificity.

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

“…In this binding mode acarbose cannot benefit from hydrogen bonding with G140H since the substrate ring at subsite -1 has no OH group. In contrast, inhibition would indicate binding at the -1 to +3 subsites, the acarbose binding mode observed in crystal structures of CGTases (24,25) (Figure 2B). …”

“…Mass spectrometry confirmed the formation of a compound with the mass of acarviosylmaltotriose -H + (808 Da) by CGTase-H140A but not by wild-type CGTase. Interestingly, to catalyze this reaction, acarbose must bind at the -2 to +2 subsites of CGTase mutant H140A, whereas it is bound in the -1 to +3 subsites of wild-type CGTase proteins according to crystal structural information (24,25), with the C-N-C linkage at the cleavage site ( Figure 2B). The CGTase structures also showed a hydrogen bond between the His140 side chain and the OH6 group of the valienamine moiety of acarbose at subsite -1, similar to the hydrogen bond formed with a natural substrate (Figure 2A), indicating the importance of this His residue for acarbose inhibition.…”

“…The PCR product (2.1 kb) was restricted with NdeI and BamHI and cloned into pET3b to yield pET3b-AT. Site-directed mutations were introduced in pET3b-AT using the QuickChange site-directed mutagenesis (5); (B) adapted from a Tabium CGTase crystal structure with a maltohexaose inhibitor (extended acarbose) (24). For clarity, not all residues at the -2, -1, and +1 subsites are shown.…”

“…ATase has the highest sequence identity (42%) with CGTases (14), and the protein is most likely organized in five domains (A-E), similar to CGTases. The substrate of ATase, acarbose, is a pseudotetrasaccharide, composed of the C7-cyclitol valienamine, 4-amino-4,6-dideoxyglucose, and maltose (Figure 3) (20)(21)(22)(23)(24)(25). In ATase, in contrast, acarbose must bind at the -2 to +2 subsites to allow catalysis of the acarviosyl transferase reaction (Figure 1).…”

“…Protein crystallography studies have shown that acarbose inhibits R-amylase family enzymes by binding in the active site with the acarviosyl moiety at the -1 and +1 subsites (20)(21)(22)(23)(24)(25). In ATase, in contrast, acarbose must bind at the -2 to +2 subsites to allow catalysis of the acarviosyl transferase reaction (Figure 1).…”