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

Jackson, Jennifer C; Duffy, Sean; Hess, Kenneth R.; Mehl, Ryan A.

doi:10.1021/ja061099y

Cited by 88 publications

(74 citation statements)

References 14 publications

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“…[55][56][57] In contrast, improving the efficiency at which an enzyme catalyzes a reaction with its native substrate is very difficult to achieve. 27,37,58 Despite the fact that paraoxon was only present in the environment for ca.…”

Section: Mechanistic Analysis Of Unnatural Phosphotriesterasesmentioning

confidence: 99%

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

Ugwumba

Ozawa

et al. 2010

J. Am. Chem. Soc.

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Abstract:The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L- (7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

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Section: Mechanistic Analysis Of Unnatural Phosphotriesterasesmentioning

confidence: 99%

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

Ugwumba

Ozawa

et al. 2010

J. Am. Chem. Soc.

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“…4). Nitroreductase mutant containing (32) at residue 124 exhibited 2-and 4-fold higher activity than even the best possible mutants consisting of only natural amino acids, F124K and F124N nitroreductase, respectively [84].…”

Section: Improvement Of Enzyme Activity and Stability By Site-specifimentioning

confidence: 93%

Manipulation of enzyme properties by noncanonical amino acid incorporation

Zheng

Kwon

2011

Biotechnology Journal

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Since wild-type enzymes do not always have the properties needed for various applications, enzymes are often engineered to obtain desirable properties through protein engineering techniques. In the past decade, complementary to the widely used rational protein design and directed evolution techniques, noncanonical amino acid incorporation (NCAAI) has become a new and effective protein engineering technique. Recently, NCAAI has been used to improve intrinsic functions of proteins, such as enzymes and fluorescent proteins, beyond the capacities obtained with natural amino acids. Herein, recent progress on improving enzyme properties through NCAAI in vivo is reviewed and the challenges of current approaches and future directions are also discussed. To date, both NCAAI methods-residue- and site-specific incorporation-have been primarily used to improve the catalytic turnover number and substrate binding affinity of enzymes. Numerous strategies used to minimize structural perturbation and stability loss of a target enzyme upon NCAAI are also explored. Considering the generality of NCAAI incorporation, we expect its application could be expanded to improve other enzyme properties, such as substrate specificity and solvent resistance in the near future.

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“…However, the orthogonality was not as high as ScGlnRS/SctRNA CUA Gln [63]. Using a semirational design strategy, Wang and Schutlz developed an excellent orthogonal pair of MjTyrRS/MjtRNA CUA Tyr , which they used to incorporate a variety of NAAs in E. coli (Table 12.2) [67][68][69][70][71]. Directed evolution was employed in which a MjtRNA CUA Tyr mutant library was generated and orthogonality was established through extensive positive and negative selection ( Figure 12.3a).…”

Section: In Vivo Ssimentioning

confidence: 99%