Insights into the "pair of sugar tongs" surface binding site in barley alpha-amylase isozymes and crystallization of appropriate sugar tongs mutants

S. Tranier, K. Deville, X. Robert, Sophie Bozonnet, R. Haser, Birte Svensson, N. Aghajari

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Recently, the three-dimensional structure of AMY1 in complex with a thio-maltotetraose (thio-DP4) has contributed to the understanding of the isozyme differences between AMY1 and AMY2, particularly the higher activity of AMY1 on starch granules. Indeed, this structure reveals the presence of an additional surface binding site called a "pair of sugar tongs" due to the sugar capturing by Tyr380 which is situated in domain C of AMYL For the first time, a biological role for the domain C was suggested as well as a hypothetical explanation of enzymatic differences between the two barley a-amylase isozymes. However, no sugar was bound at the "sugar tongs" site in the AMY2/acarbose complex. Comparative studies of this domain on the basis of sequence, secondary structure and spatial organization allow to propose factors needed for such a site. One of the most obvious is the replacement of Ser378(AMY1) by Prc,376(AMY2), which could be responsible for the lack of the surface binding site by inducing a more rigid structure of the loop preceding Tyr380. In order to test this hypothesis, two mutants of this particular residue have been engineered: AMY1(S378P) mimicking the AMY2 and AMY1(S378T). However, surface plasmon resonance sugar binding experiments have proven unambiguously that this residue cannot totally explain the lack of the "pair of sugar tongs" and other tracks must be studied as, for example, the differences in orientation of Asp381 and the critical role of His395, both good candidates for mutational engineering. Also, the contribution of domain A could be suggested in addition to minor differences implying interacting residues at the "sugar tongs" binding site. Finally, the successful crystallization of five mutants in the "sugar tongs" binding site is reported.
    Original languageEnglish
    JournalBiologia
    Volume60
    Pages (from-to)37-46
    ISSN0006-3088
    Publication statusPublished - 2005

    Keywords

    • "a pair of sugar tongs"
    • barley
    • surface binding sites
    • crystallization
    • alpha-amylase mutants
    • isozymes

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