Crystal Structure and Substrate Enantioselectivity of-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase
Date Issued
2009
Date
2009
Author(s)
Chou, Chien-Yu
Abstract
L-form peptides that bind specifically to L-sugars can be prepared by phage display. The corresponding D-form peptides which can be synthesized chemically can then be used to target natural D-form sugars on cell-surface. Such D-form peptides may be developed as drug candidates for various diseases. The strategy requires the unnatural, enantiomeric L-sugars. In a recent directed-evolution study, Escherichia coli D-sialic acid aldolase was converted by introducing eight point mutations into-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase which showed a preferred enantioselectivity toward L-sugars. In this study, the crystal structures of L-KDO aldolase at 1.98-Å resolution and D-sialic acid aldolase at 1.47-Å resolution have been determined in order to investigate the underlying molecular basis of substratenantioselectivity. Both enzymes are tetramers with each subunit consisting of a (α/β)8 barrel. The structural information indicated that all mutations are away from theatalytic center, except for V251I which is near the opening of the (α/β)8-barrel and is proximal to the Schiff base-forming Lys165. The V→I substitution causes theugar-binding pocket to become relatively narrow, thus creating a large steric hindrance for the sugar binding. The crystal structure of L-KDO aldolase in complex withydroxypyruvate (a product analogue) has also been solved to allow the precise docking of the substrates L-KDO and D-sialic acid into the active site. It is interesting that only L-KDO can be properly accommodated in the narrow binding pocket. Moreover, the crystal structure of D-sialic acid aldolase in complex with L-arabinose was determined, which points out that the wide sugar-binding pocket may allow the product L-arabinose to slip into the neighboring cavity during catalysis of the L-KDO cleavage, resulting in a decreased cleavage activity. Further enzymatic and structural analyses indicated that single mutations on V251 are sufficient to invert the enantioselectivity toward L-sugar, in good agreement with the structural observations described above. Based on theseesults, we propose that the substrate specificity of sialic acid / KDO aldolase can be regulated by changing the size of sugar-binding pocket.
Subjects
L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase
Substrate enantioselectivity
Directed evolution
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