Journal
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
Volume 79, Issue 2, Pages 633-644Publisher
WILEY
DOI: 10.1002/prot.22911
Keywords
amylomaltase; 250s loop; substrate binding sites; Thermus brockianus
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Funding
- National Research Foundation [2010-0011602]
- KRIBB
- National Research Foundation of Korea [2010-0011602] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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Amylomaltase, or 4-alpha-glucanotransferase (EC 2.4.1.25), is involved in glycogen and maltooligosaccharide metabolism in microorganisms, catalyzing both the hydrolysis and transfer of an alpha-1,4-oligosacchraride to other sugar molecules. In this study, we determined the crystal structure of amylomaltase from Thermus brockianus at a resolution of 2.3 angstrom and conducted a biochemical study to understand the detailed mechanism for its activity. Careful comparison with previous amylomaltase structures showed a pattern of conformational flexibility in the 250s loop with higher B-factor. Amylomaltase from T. brockianus exhibited a high transglycosylation factor for glucose and a lower value for maltose. Mutation of Gln256 resulted in increased K-m for maltotriose and a sharp decrease of the transglycosylation factor for maltose, suggesting the involvement of Gin 256 in substrate binding between subsites +1 and +2. Mutation of Phe251 resulted in significantly lower glucose production but increased maltose production from maltopentose substrates, showing an altered substrate-binding affinity. The mutational data suggest the conformational flexibility of the loop may be involved in substrate binding in the GH77 family. Here, we present an action model of the 250s loop providing the molecular basis for the involvement of residues Phe251, Gln256, and Trp258 in the hydrolysis and transglycosylation activities in amylomaltase. Proteins 2011; 79:633-644. (C) 2010 Wiley-Liss, Inc.
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