Enzymes in carbohydrate synthesis:  N-acetylneuraminic acid aldolase catalyzed reactions and preparation of N-acetyl-2-deoxy-D-neuraminic acid derivatives

Kim, Mahn Joo; Hennen, William J.; Sweers, H. M.; Wong, Chi Huey

doi:10.1021/ja00227a031

Cited by 200 publications

(82 citation statements)

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“…NAL is highly specific for its ketone donor (pyruvate) while accepting a range of aldehyde acceptors (38,39). However, the wild-type enzyme has a strong preference for longer aldehydes (38)(39)(40): C4 aldehydes are generally poorly accepted (38) and C3 aldehydes are not substrates (39). The differently modified enzymes at each of the positions in the polypeptide chain were screened in 96-well plates for altered activity and specificity in condensing pyruvate with a range of aldehyde substrates with different lengths (C4 to C6), stereochemistry, and functional groups (Fig.…”

Section: Resultsmentioning

confidence: 99%

Extending enzyme molecular recognition with an expanded amino acid alphabet

Windle

Simmons

Ault

et al. 2017

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

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Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties.protein engineering | aldolases | chemical modification E nzymes are phenomenally powerful catalysts that increase reaction rates by up to 10 18 -fold (1, 2), and a new era of enzyme applications has been opened by the advancement of protein engineering and directed evolution to provide new, or improved, enzymes for industrial biocatalysis. Enzymes are attractive catalysts because they are highly selective, carrying out regio-, chemo-, and stereoselective reactions that are challenging for conventional chemistry. Moreover, enzymes are efficient catalysts, function under mild conditions with relatively nontoxic reagents, and enable the production of relatively pure products, minimizing waste generation. In recent years, there has been much success in engineering enzymes for desired reactions (3-5) using methods such as rational protein engineering (6-8), directed evolution (9-11), and, most recently, computational enzyme design (12-15).Enzymes found in Nature achieve catalysis using active sites generally composed of only 20 canonical amino acids, which are encoded at the genetic level, plus the rarer selenocysteine and 1-pyrrolysine. However, many enzymes also rely on one or more of 27 small organic cofactor molecules and/or 13 metal ions for their function (16). In addition, in some cases, Nature has exploited noncanonical amino acids (Ncas) in catalysis to extend its catalytic repertoire: for example, the quinones TPQ, LTQ, TTQ, and CTQ, respectively, in ...

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Section: Resultsmentioning

confidence: 99%

Extending enzyme molecular recognition with an expanded amino acid alphabet

Windle

Simmons

Ault

et al. 2017

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

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“…By coupling of the GlcNAc 2-epimerase (AGE, EC 5.3.1.8) with NanA, a 2-step enzymatic approach was developed for Neu5Ac production (21,22,41). This procedure is regarded as the most cost-effective enzymatic method for Neu5Ac production on an (17,18,24,25). Further improvement of its application potential, by simultaneously displaying GlcNAc 2-epimerase and NanA on the spore surface, represents a new strategy for the enzymatic production of Neu5Ac.…”

Section: Discussionmentioning

confidence: 99%

Production of N -Acetyl- d -Neuraminic Acid by Use of an Efficient Spore Surface Display System

Gao

Zhang

et al. 2011

Appl Environ Microbiol

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Production of N-acetyl-D-neuraminic acid (Neu5Ac) via biocatalysis is traditionally conducted using isolated enzymes or whole cells. The use of isolated enzymes is restricted by the time-consuming purification process, whereas the application of whole cells is limited by the permeability barrier presented by the microbial cell membrane. In this study, a novel type of biocatalyst, Neu5Ac aldolase presented on the surface of Bacillus subtilis spores, was used for the production of Neu5Ac. Under optimal conditions, Neu5Ac at a high concentration (54.7 g liter ؊1 ) and a high yield (90.2%) was obtained under a 5-fold excess of pyruvate over N-acetyl-D-mannosamine. The novel biocatalyst system, which is able to express and immobilize the target enzyme simultaneously on the surface of B. subtilis spores, represents a suitable alternative for value-added chemical production.

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“…The enzymatic synthesis of NeuAc from Nacetyl-D-mannosamine (ManNAc) and pyruvate with N-acetylneuraminate pyurvate lyase (Neu5Ac adolase, NAL, EC 4.1.3.3) has been reported (Augé et al, 1984;Kim et al, 1988). Chemoenzymatic processes, incorporating chemical epimerization of N-acetyl-D-glucosamine (GlcNAc) to ManNAc under alkaline conditions and biotransformation of ManNAc to NeuAc with NAL, that allow the use of the relatively inexpensive GlcNAc as the starting material have also been developed (Simon et al, 1988).…”

Section: Introductionmentioning

confidence: 99%