Multiple mutations in the aglycone binding pocket to convert the substrate specificity of dalcochinase to linamarase
- Department of Biochemistry, Faculty of Science, and Center for Advanced Studies in Tropical Natural Resources, NRU-KU, Kasetsart University, 50 Paholyothin Road, Chatuchak, Bangkok, 10900 (Thailand)
- Laboratory of Biochemistry, Chulabhorn Research Institute, 54 Kamphaeng Phet 6 Road, Laksi, Bangkok, 10210 (Thailand)
Highlights: • Dalcochinase and linamarase are β-glucosidases with distinct substrate specificities. • Substrate specificities could be converted by substitution of 2–3 key residues. • The I185A mutation is needed to fit substrates with tri-functional aglycone moieties. • The N189F mutant showed a potential for enzymatic synthesis of alkyl glucosides. • β-Glucosidases could be tailored to suit desired hydrolytic or synthetic applications. Dalcochinase from Dalbergia cochinchinensis Pierre and linamarase from Manihot esculenta Crantz are β-glucosidases which share 47% sequence identity, but show distinct substrate specificities in hydrolysis and transglucosylation. Previously, three amino acid residues of dalcochinase, namely I185, N189 and V255, were identified as being important for determining substrate specificity. In this study, kinetic analysis of the ensuing double and triple mutants of dalcochinase showed that only those containing the 185A mutation could appreciably hydrolyze linamarin as well as transfer glucose to 2-methyl-2-propanol. So, the space provided by the I185A mutation appeared to be a prerequisite for accommodation of the aglycone moiety containing three substituents at the carbinol carbon. However, quantitative analysis of the energy parameters revealed mostly antagonistic interactions between these mutations. In addition, the N189F mutant showed a potential for use in enzymatic synthesis of alkyl glucosides via transglucosylation and reverse hydrolysis reactions. Thus, substitution of only 2–3 key residues in the aglycone binding pocket of dalcochinase could convert its specificities to that of linamarase, as well as to be suitable for any chosen hydrolytic or synthetic applications.
- OSTI ID:
- 23134168
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 504, Issue 4; Other Information: Copyright (c) 2018 Elsevier Inc. All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
Similar Records
Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development
Novel {alpha}-glucosidase from human gut microbiome : substrate specificities and their switch.