Chemical modification in the creation of novel biocatalysts

Díaz‐Rodríguez, Alba; Davis, Benjamin G.

doi:10.1016/j.cbpa.2010.12.002

Cited by 101 publications

(67 citation statements)

References 44 publications

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“…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 amine oxidase, lysyl oxidase, methylamine dehydrogenase, and quinohemoprotein amine dehydrogenase (17); a pyruvoyl group in some histidine, arginine, aspartate, and Sadenosylmethionine decarboxylases (18); formyl glycine residues in type I sulfatases (19); and 4-methylideneimidazole-5-one (MIO) in ammonia lyases and 2,3-aminomutases (20). These Ncas are vital for catalysis and arise through posttranslational modifications of the polypeptide chain (21, 22), allowing access to chemistries not otherwise provided by the 20 proteogenic amino acids.Technologies for the protein engineer to incorporate Ncas into proteins at specifically chosen sites, either by genetic means (23)(24)(25) or by chemical modification (26,27), have recently been developed. These approaches are powerful because, unlike traditional protein engineering with the 20 canonical amino acids, protein engineering with Ncas has almost unlimited novel side-chain structures and chemistries from which to choose.…”

mentioning

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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mentioning

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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“…This strategy has been used in many instances to increase the solubility of the enzymes in some unconventional media; e.g., the hydrophobization of the enzyme surface may produce an enzyme composite that was soluble in organic media [3][4][5][6]. Thus, in some instances the enzyme surface has been modified with polyethyleneglycol (PEG) and used in organic medium, improving the enzyme solubility in this organic medium, and in that way its activity [7][8][9] (otherwise, the enzyme will be in an aggregated form leading to diffusional limitations) [10].…”

Section: Introductionmentioning

confidence: 99%

Solid-phase modification with succinic polyethyleneglycol of aminated lipase B from Candida antarctica: Effect of the immobilization protocol on enzyme catalytic properties

Ruiz

Galvis

Barbosa

et al. 2013

Journal of Molecular Catalysis B: Enzymatic

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“…However, there is more than one reactive group in BMP2, and this method might suffer from poor site-specificity and heterogeneous products. In addition, non-specific chemical modifications can result in inactivation of proteins or their incorrect orientation 18–21 .…”

Section: Introductionmentioning

confidence: 99%

BMP2-modified injectable hydrogel for osteogenic differentiation of human periodontal ligament stem cells

Park

Kwon

Lee

et al. 2017

Sci Rep

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This is the first report on the development of a covalently bone morphogenetic protein-2 (BMP2)-immobilized hydrogel that is suitable for osteogenic differentiation of human periodontal ligament stem cells (hPLSCs). O-propargyl-tyrosine (OpgY) was site-specifically incorporated into BMP2 to prepare BMP2-OpgY with an alkyne group. The engineered BMP2-OpgY exhibited osteogenic characteristics after in vitro osteogenic differentiation of hPLSCs, indicating the osteogenic ability of BMP2-OpgY. A methoxy polyethylene glycol-(polycaprolactone-(N3)) block copolymer (MC-N3) was prepared as an injectable in situ-forming hydrogel. BMP2 covalently immobilized on an MC hydrogel (MC-BMP2) was prepared quantitatively by a simple biorthogonal reaction between alkyne groups on BMP2-OpgY and azide groups on MC-N3 via a Cu(I)-catalyzed click reaction. The hPLSCs-loaded MC-BMP2 formed a hydrogel almost immediately upon injection into animals. In vivo osteogenic differentiation of hPLSCs in the MC-BMP2 formulation was confirmed by histological staining and gene expression analyses. Histological staining of hPLSC-loaded MC-BMP2 implants showed evidence of mineralized calcium deposits, whereas hPLSC-loaded MC-Cl or BMP2-OpgY mixed with MC-Cl, implants showed no mineral deposits. Additionally, MC-BMP2 induced higher levels of osteogenic gene expression in hPLSCs than in other groups. In conclusion, BMP2-OpgY covalently immobilized on MC-BMP2 induced osteogenic differentiation of hPLSCs as a noninvasive method for bone tissue engineering.

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Chemical modification in the creation of novel biocatalysts

Cited by 101 publications

References 44 publications

Extending enzyme molecular recognition with an expanded amino acid alphabet

Extending enzyme molecular recognition with an expanded amino acid alphabet

Solid-phase modification with succinic polyethyleneglycol of aminated lipase B from Candida antarctica: Effect of the immobilization protocol on enzyme catalytic properties

BMP2-modified injectable hydrogel for osteogenic differentiation of human periodontal ligament stem cells

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