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

Research output: Contribution to journalArticlepeer-review

10 Citations (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.

Original languageEnglish
Pages (from-to)14057-14064
Number of pages8
JournalJournal of Biological Chemistry
Issue number16
Publication statusPublished - Apr 22 2011
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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