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Kragl, Udo; Gödde, Astrid; Wandrey, Christian; Lubin, Nadège; Augé, Claudine; Scobie, Martin; Mahon, Mary F.; Threadgill, Michael D.; Guillerm, Georges; Gâtel, Marie

doi:10.1039/p19940000909

Cited by 6 publications

(15 citation statements)

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“…Although enzyme adaptation for substrate analogues is unpredictable (19), it has been demonstrated that activities of some substrate analogues do increase throughout the course of directed evolution (10,14,20). The wild-type sialic acid aldolase exhibits a low level of activity toward D-and L-KDO (5,21). By alternating D-and L-KDO as a starting substrate in screening, we have selected several mutants with improvements in KDO catalysis (11).…”

Section: Resultsmentioning

confidence: 99%

“…D-sialic acid is an essential cell-surface residue associated with inflammatory diseases, cancer metastasis, and influenza virus infection (3,4). A number of sialic acid analogs have been synthesized for their numerous biological and pharmaceutical applications (5,6). The structure of the sialic acid aldolase is composed of eight repeating ␣͞␤ barrels, (7) representing the most common structural motif for aldolases (8).…”

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confidence: 99%

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Directed evolution of d -sialic acid aldolase to l -3-deoxy- manno -2-octulosonic acid ( l -KDO) aldolase

Hsu

Hong

Wada

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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An efficient L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase was created by directed evolution from the Escherichia coli D-Neu5Ac (N-acetylneuraminic acid, D-sialic acid) aldolase. Five rounds of error-prone PCR and iterative screening were performed with sampling of 10 3 colonies per round. The specificity constant (kcat͞Km) of the unnatural sugar L-KDO is improved to a level equivalent to the wild-type D-sialic acid aldolase for its natural substrate, D-Neu5Ac. The final evolved enzyme exhibits a >1,000-fold improved ratio of the specificity constant [kcat͞Km (L-KDO)]͞ [kcat͞Km (D-sialic acid)]. The protein sequence of the evolved aldolase showed eight amino acid changes from the native enzyme, with all of the observed changes occurring outside of the active site. Our effort demonstrates that an enzyme can be rapidly altered to accept enantiomeric substrates with screening of a small population of colonies iteratively toward the target substrate with improved catalytic efficiency. This work provides a method for the synthesis of enantiomeric sugars and for the study of enantiomeric catalysis affected by remote mutations.inversion of enanselectivity ͉ enzyme engineering ͉ L-sugar synthesis C arbohydrates constitute unique cellular structures that play a vital role in various molecular recognition processes. Interference of this recognition process presents a great potential in biomedical and pharmaceutical applications. Thus, developing methods of interference-targeting cell-surface carbohydrates may offer therapeutic candidates for treatment of inflammation, cancer metastasis, and bacterial or viral infection (1). Previously, we developed a strategy in which enantiomers of natural peptides are identified to target cell-surface sugars through in vitro phage selection (2) (Fig. 1). These D-peptide binders of cell-surface carbohydrates provide a valuable tool for the study of protein and carbohydrate interactions. With this approach, we have identified a dodecapeptide binding to sialic acid with a dissociation constant (K d ) of Ϸ0.5 mM. Another target of interest is D-3-deoxy-manno-2-octulosonic acid (D-KDO), which is an essential component of the outer-cell membrane lipopolysaccharide of Gram-negative bacteria. These Dpeptide binders of D-KDO may offer previously unrecognized antibacterial agents. A prerequisite to this approach is an efficient synthesis of the enantiomeric L-sugars of the natural D-configured carbohydrates. The current preparative use of the wild-type N-acetylneuraminic acid aldolase (sialic acid aldolase, EC 4.1.3.3) for the synthesis of L-sialic acid and other highcarbon L-sugars is hampered by its low turnover rate; therefore, improving sialic acid aldolase toward accepting the unnatural L-enantiomeric substrate is needed to facilitate its application in synthetic chemistry.N-acetyl-D-neuraminic acid aldolase (sialic acid aldolase) catalyses the reversible aldol reaction of N-acetyl-D-mannosamine and pyruvate to produce N-acetyl-D-neuraminic acid (D-sialic acid) (Fig. 2). D-sialic acid ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Directed evolution of d -sialic acid aldolase to l -3-deoxy- manno -2-octulosonic acid ( l -KDO) aldolase

Hsu

Hong

Wada

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Several explanations for the stereoselectivity of NAL have been proposed. 9,12,13 In particular, Wong et al 9 have suggested that the acceptor binds the enzyme in a boatlike conformation when re-face attack occurs and in a chairlike conformation for si-face attack. This enables attack of the acceptor aldehyde carbonyl from the same face of the enamine but with different faces of the aldehyde group.…”

Section: Introductionmentioning

confidence: 99%

Substrate-Assisted Catalysis in Sialic Acid Aldolase

Smith¹,

Lawrence²,

Barbosa³

1999

J. Org. Chem.

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Sialic acid aldolase catalyses the reversible aldol condensation of pyruvate and N-acetylmannosamine with an apparent lack of stereospecificity. Consistent with this, modeling of Schiff base and enamine intermediates in the active site of this enzyme yields two conformations, corresponding to si- and re-face attack in the aldol condensation reaction. The acceptor-aldehyde group is found on different sides of the enamine in the two conformations, but with the remainder of the substrate having very similar geometries in the protein. No histidine residue previously speculated to function as a general base in the mechanism is found near the enzyme active site. In the absence of functionally active groups in the active site, the carboxylate of the substrate is proposed to function as the general acid/base. Molecular orbital calculations indicate that the barrier to aldol cleavage via this mechanism in the gas phase of the related system, 4-hydroxy-2-methyiminopentanoic acid, is 74 kJ mol(-)(1).

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“…Purification of our KDO product using Dowex 1 × 8 (200-400 mesh) anion-exchange chromatography following the method reported by Kragl et al,20 resulted in separation of KDO from 4-epi-KDO, allowing complete assignment of their respective 1 H NMR spectra (see the Supporting Information).…”

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confidence: 99%

“…In this way we, as well as others, 1a,9a,b have observed that the stereochemical outcome for the condensation between arabinose and oxalacetic acid appears to favour the formation of KDO over 4-epi-KDO, with a ratio of KDO to 4epi-KDO typically around 4:1. Purification of our KDO product using Dowex 1 × 8 (200-400 mesh) anion-exchange chromatography following the method reported by Kragl et al, 20 resulted in separation of KDO from 4epi-KDO, allowing complete assignment of their respective 1 H NMR spectra (see the Supporting Information).…”

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confidence: 99%