Modulation of substrate specificities of D-sialic acid aldolase through single mutations of val-251

Chien Yu Chou, Tzu Ping Ko, Kuan Jung Wu, Kai Fa Huang, Chun Hung Lin, Chi Huey Wong, Andrew H.J. Wang

研究成果: 雜誌貢獻文章同行評審

10 引文 斯高帕斯(Scopus)


In a recent directed-evolution study, Escherichia coli D-sialic acid aldolase was converted by introducing eight point mutations into a new enzyme with relaxed specificity, denoted RS-aldolase (also known formerly as L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase), which showed a preferred selectivity toward L-KDO. To investigate the underlying molecular basis, we determined the crystal structures of D-sialic acid aldolase and RS-aldolase. All mutations are away from the catalytic center, except for V251I, which is near the opening of the (α/β)8-barrel and proximal to the Schiff base-forming Lys-165. The change of specificity from D-sialic acid to RS-aldolase can be attributed mainly to the V251I substitution, which creates a narrower sugar-binding pocket, but without altering the chirality in the reaction center. The crystal structures of D-sialic acid aldolase-L-arabinose and RS-aldolase-hydroxypyruvate complexes and five mutants (V251I, V251L, V251R, V251W, and V251I/V265I) of the D-sialic acid aldolase were also determined, revealing the location of substrate molecules and how the contour of the active site pocket was shaped. Interestingly, by mutating Val251 alone, the enzyme can accept substrates of varying size in the aldolase reactions and still retain stereoselectivity. The engineered D-sialic acid aldolase may find applications in synthesizing unnatural sugars of C6 to C10 for the design of antagonists and inhibitors of glycoenzymes.

頁(從 - 到)14057-14064
期刊Journal of Biological Chemistry
出版狀態已發佈 - 4月 22 2011

ASJC Scopus subject areas

  • 生物化學
  • 分子生物學
  • 細胞生物學


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