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Extending enzyme molecular recognition with an expanded amino acid alphabet

DOI: 10.1073/pnas.1616816114 DOI Help

Authors: Claire L. Windle (Astbury Centre for Structural Molecular Biology; School of Molecular and Cellular Biology, University of Leeds) , Katie J. Simmons (Astbury Centre for Structural Molecular Biology; School of Chemistry, University of Leeds) , James R. Ault (Astbury Centre for Structural Molecular Biology; School of Molecular and Cellular Biology, University of Leeds) , Chi H. Trinh (Astbury Centre for Structural Molecular Biology; School of Molecular and Cellular Biology, University of Leeds) , Adam Nelson (Astbury Centre for Structural Molecular Biology; School of Chemistry, University of Leeds) , Arwen R. Pearson (Astbury Centre for Structural Molecular Biology, University of Leeds) , Alan Berry (Astbury Centre for Structural Molecular Biology; School of Molecular and Cellular Biology, University of Leeds)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences

State: Published (Approved)
Published: February 2017
Diamond Proposal Number(s): 10305

Abstract: 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.

Journal Keywords: protein engineering; aldolases; chemical modification

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)