Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity

Wright, Tom H.; Bower, B.; Chalker, Justin M.; Bernardes, Gonçalo J. L.; Wiewiora, Rafal; Ng, Wai-Lung; Raj, Ritu; Faulkner, Sarah; Vallée, M. Robert J.; Phanumartwiwath, Anuchit; Coleman, Oliver D.; Thézénas, Marie‐Laëtitia; Khan, M. Adil; Galan, Sébastien R. G.; Lercher, Lukas; Schombs, Matthew W.; Gerstberger, Stefanie; Palm-Espling, Maria E.; Baldwin, Andrew J.; Kessler, Benedikt M.; Claridge, Timothy D. W.; Mohammed, Shabaz; Davis, Benjamin G.

doi:10.1126/science.aag1465

Cited by 268 publications

(257 citation statements)

References 105 publications

(130 reference statements)

Supporting

Mentioning

244

Contrasting

Unclassified

Order By: Relevance

“…In stark contrast, none of the canonical amino acids possess the required molecular characteristics in a single residue, demonstrating that Ncas can extend the ambitions of the protein engineer. Based on the data presented here, we envisage that advances in computational (12,13) and (semi)rational methods (6) for enzyme redesign, coupled with the development of recent powerful chemical mutagenesis methods (51) and the enormous variety of Ncas side chains and chemistries, will open the way to engineer enzymes with catalytic functions that are not found in Nature.…”

Section: Discussionmentioning

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.

View full text Add to dashboard Cite

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 ...

show abstract

Section: Discussionmentioning

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.

View full text Add to dashboard Cite

show abstract

“…1 A handful of reaction classes are well-suited to fulfill these requirements, including the Staudinger ligation, 2 1,3-dipolar cycloadditions of azides, 3,4 diazo compounds, 5 nitrones 6 or nitrile imines, 7 oxime or hydrazone formation, 8 esterifications, 5g,9 tetrazine ligations, 10 and others. 11 …”

Section: Introductionmentioning

confidence: 99%

Fine-Tuning Strain and Electronic Activation of Strain-Promoted 1,3-Dipolar Cycloadditions with Endocyclic Sulfamates in SNO-OCTs

et al. 2017

View full text Add to dashboard Cite

The ability to achieve predictable control over the polarization of strained cycloalkynes can influence their behavior in subsequent reactions, providing opportunities to increase both rate and chemoselectivity. A series of new heterocyclic strained cyclooctynes containing a sulfamate backbone (SNO-OCTs) were prepared under mild conditions by employing ring expansions of silylated methyleneaziridines. SNO-OCT derivative 8 outpaced even a difluorinated cyclooctyne in a 1,3-dipolar cycloaddition with benzylazide. The various orbital interactions of the propargylic and homopropargylic heteroatoms in SNO-OCT were explored both experimentally and computationally. The inclusion of these heteroatoms had a positive impact on stability and reactivity, where electronic effects could be utilized to relieve ring strain. The choice of the heteroatom combinations in various SNO-OCTs significantly affected the alkyne geometries, thus illustrating a new strategy for modulating strain via remote substituents. Additionally, this unique heteroatom activation was capable of accelerating the rate of reaction of SNO-OCT with diazoacetamide over azidoacetamide, opening the possibility of further method development in the context of chemoselective, bioorthogonal labeling.

show abstract

“…14 Unlike the canonical twenty amino acids, dehydroalanine is electrophilic, making it suitable for selective functionalization even within the complex environment of a natural product, peptide, or protein. 15,16 As such, a strategy for catalytic, nucleophilic addition to dehydroalanines in a complex environment would enable post-synthetic, catalyst-controlled chemical editing of many naturally-occurring compounds (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

Site- and Stereoselective Chemical Editing of Thiostrepton by Rh-Catalyzed Conjugate Arylation: New Analogues and Collateral Enantioselective Synthesis of Amino Acids

Key

Miller

2017

J. Am. Chem. Soc.

View full text Add to dashboard Cite

The synthesis of complex, biologically active molecules by catalyst-controlled, selective functionalization of complex molecules is an emerging capability. We describe the application of Rh-catalyzed conjugate arylation to the modification of thiostrepton, a complex molecule with potent antibacterial properties for which few analogs are known. By this approach, we achieve the site- and stereoselective functionalization of one subterminal dehydroalanine residue (Dha16) present in thiostrepton. The broad scope of this method enabled the preparation and isolation of 24 new analogs of thiostrepton, the biological testing of which revealed that the antimicrobial activity of thiostrepton tolerates the alternation of Dha16 to a range of amino acids. Further analysis of this Rh-catalyzed process revealed that use of sodium or potassium salts was crucial for achieving high stereoselectivity. The catalyst system was studied further by application to the synthesis of amino esters and amides from dehydroalanine monomers, a process which was found to occur with up to 93 : 7 er under conditions milder than those previously reported for analogous reactions. Furthermore, the addition of the same sodium and potassium salts as applied in the case of thiostrepton lead to a nearly full reversal of the enantioselectivity of the reaction. As such, this study of site-selective catalysis in a complex molecular setting also delivered synergistic insights in the arena of enantioselective catalysis. In addition, these studies greatly expand the number of known thiostrepton analogs obtained by any method and reveal a high level of functional group tolerance for metal-catalyzed, site-selective modifications of highly complex natural products.

show abstract

Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity

Cited by 268 publications

References 105 publications

Extending enzyme molecular recognition with an expanded amino acid alphabet

Extending enzyme molecular recognition with an expanded amino acid alphabet

Fine-Tuning Strain and Electronic Activation of Strain-Promoted 1,3-Dipolar Cycloadditions with Endocyclic Sulfamates in SNO-OCTs

Site- and Stereoselective Chemical Editing of Thiostrepton by Rh-Catalyzed Conjugate Arylation: New Analogues and Collateral Enantioselective Synthesis of Amino Acids

Contact Info

Product

Resources

About