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

ABSTRACT: 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 (?/?)(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 C(6) to C(10) for the design of antagonists and inhibitors of glycoenzymes.

SUBMITTER: Chou CY

PROVIDER: S-EPMC3077606 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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

Chou Chien-Yu CY Ko Tzu-Ping TP Wu Kuan-Jung KJ Huang Kai-Fa KF Lin Chun-Hung CH Wong Chi-Huey CH Wang Andrew H-J AH

The Journal of biological chemistry 20110126 16

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, e ...[more]

PMID: 21270125

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Dataset Information

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

Publications

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

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