Improving Nature's Enzyme Active Site with Genetically Encoded Unnatural Amino Acids

2006 ◽  
Vol 128 (34) ◽  
pp. 11124-11127 ◽  
Author(s):  
Jennifer C. Jackson ◽  
Sean P. Duffy ◽  
Kenneth R. Hess ◽  
Ryan A. Mehl
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Unnatural Amino Acids ◽  
Enzyme Active Site

Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 3028-3034 ◽  
Author(s):  
Soohee Lee ◽  
Asim K. Debnath ◽  
Colvin M. Redman
Keyword(s):  
Amino Acids ◽  
Blood Group ◽  
Active Site ◽  
Neutral Endopeptidase ◽  
Site Directed Mutagenesis ◽  
Peptide Hydrolysis ◽  
Enzyme Active Site ◽  
Endopeptidase 24.11 ◽  
Outer Domain ◽  
Group Protein

Abstract In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3–converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of α-helical segments. The domain situated closest to the membrane contains both the N- and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. Our studies indicate that Kell and NEP use the same homologous amino acids in the coordination of zinc and in peptide hydrolysis. However, Kell uses different amino acids than NEP in substrate binding and appears to have more flexibility in the composition of amino acids allowed in the active site.

scholarly journals Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

2015 ◽  
Vol 81 (20) ◽  
pp. 6994-7002 ◽  
Author(s):  
Sang-Woo Han ◽  
Eul-Soo Park ◽  
Joo-Young Dong ◽  
Jong-Shik Shin
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Unnatural Amino Acids ◽  
Kinetic Resolution ◽  
Enantiomeric Excess ◽  
Active Site Residues ◽  
Alanine Scanning Mutagenesis ◽  
Content Type ◽  
Keto Acids ◽  
Steric Constraint

ABSTRACTω-Transaminase (ω-TA) is a promising enzyme for use in the production of unnatural amino acids from keto acids using cheap amino donors such as isopropylamine. The small substrate-binding pocket of most ω-TAs permits entry of substituents no larger than an ethyl group, which presents a significant challenge to the preparation of structurally diverse unnatural amino acids. Here we report on the engineering of an (S)-selective ω-TA fromOchrobactrum anthropi(OATA) to reduce the steric constraint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in whichl-alanine was used as a ligand, nine active-site residues were selected for alanine scanning mutagenesis. Among the resulting variants, an L57A variant showed dramatic activity improvements in activity for α-keto acids and α-amino acids carrying substituents whose bulk is up to that of ann-butyl substituent (e.g., 48- and 56-fold increases in activity for 2-oxopentanoic acid andl-norvaline, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In contrast, an L57V substitution failed to induce the improvements in activity for bulky substrates. Molecular modeling suggested that the alanine substitution of L57, located in a large pocket, induces an altered binding orientation of an α-carboxyl group and thereby provides more room to the small pocket. The synthetic utility of the L57A variant was demonstrated by carrying out the production of optically purel- andd-norvaline (i.e., enantiomeric excess [ee] > 99%) by asymmetric amination of 2-oxopantanoic acid and kinetic resolution of racemic norvaline, respectively.

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