Complex Structures of Thermoactinomyces vulgaris R-47 α-Amylase 2 with Acarbose and Cyclodextrins Demonstrate the Multiple Substrate Recognition Mechanism

Abstract

Thermoactinomyces vulgaris R-47 ␣-amylase 2 (TVAII) has the unique ability to hydrolyze cyclodextrins (CDs), with various sized cavities, as well as starch. To understand the relationship between structure and substrate specificity, x-ray structures of a TVAII-acarbose complex and inactive mutant TVAII (D325N/D421N)/␣-, ␤-and ␥-CDs complexes were determined at resolutions of 2.9, 2.9, 2.8, and 3.1 Å, respectively. In all complexes, the interactions between ligands and enzymes at subsites ؊1, ؊2, and ؊3 were almost the same, but striking differences in the catalytic site structure were found at subsites ؉1 and ؉2, where Trp 356 and Tyr 374 changed the conformation of the side chain depending on the structure and size of the ligands. Trp 356 and Tyr 374 are thought to be responsible for the multiple substraterecognition mechanism of TVAII, providing the unique substrate specificity. In the ␤-CD complex, the ␤-CD maintains a regular conical structure, making it difficult for Glu 354 to protonate the O-4 atom at the hydrolyzing site as a previously proposed hydrolyzing mechanism of ␣-amylase. From the x-ray structures, it is suggested that the protonation of the O-4 atom is possibly carried out via a hydrogen atom of the inter-glucose hydrogen bond at the hydrolyzing site.

Structural insights into the catalytic reaction that is involved in the reorientation of Trp238 at the substrate‐binding site in GH13 dextran glucosidase

“…Halothermothrix orenii a-amylase (GH13_36) [22][23][24][25][26][27]. Two orientation patterns of the Trp side-chain are observed in GH13 enzymes with known structures.…”

Changes in the Catalytic Properties of Pyrococcus furiosus Thermostable Amylase by Mutagenesis of the Substrate Binding Sites

“…1. According to the structures of GH-13 enzymes bound to their substrates, the last two residues of the second conserved region (Lys and His in ␣-amylases and CGTases and Asn and Glu in CD-degrading enzymes) and a hydrophobic residue in the third conserved region (Trp in ␣-amylases and CD-degrading enzymes and Phe in CGTases) play important roles in substrate binding to the ϩ1 and ϩ2 subsites (16,25). As shown in Fig.…”

Structural Elucidation of Dextran Degradation Mechanism by Streptococcus mutans Dextranase Belonging to Glycoside Hydrolase Family 66

“…The catalytic nucleophile is the aspartic acid located at the end of the (␤/␣) 8 -barrel fourth ␤-strand, and the proton donor is the glutamic acid located at the fifth ␤-strand end (Fig. 5B) (44,58).…”