Skip to main content

Improving a natural enzyme activity through incorporation of unnatural amino acids

Journal Article


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.

Authors


  •   Ugwumba, Isaac N. (external author)
  •   Ozawa, Kiyoshi (external author)
  •   Xu, Zhi-Qiang
  •   Ely, Fernanda (external author)
  •   Foo, Jee-Loon (external author)
  •   Herlt, Anthony J. (external author)
  •   Coppin, Chris (external author)
  •   Brown, Sue (external author)
  •   Taylor, Matthew C. (external author)
  •   Ollis, David L. (external author)
  •   Mander, Lewis N. (external author)
  •   Schenk, Gerhard (external author)
  •   Dixon, Nicholas E.
  •   Otting, Gottfried (external author)
  •   Oakeshott, John G. (external author)
  •   Jackson, Colin J. (external author)

Publication Date


  • 2011

Citation


  • Ugwumba, I. N., Ozawa, K., Xu, Z., Ely, F., Foo, J., Herlt, A. J., Coppin, C., Brown, S., Taylor, M. C., Ollis, D. L., Mander, L. N., Schenk, G., Dixon, N. E., Otting, G., Oakeshott, J. G. & Jackson, C. J. (2011). Improving a natural enzyme activity through incorporation of unnatural amino acids. Journal of the American Chemical Society, 133 (2), 326-333.

Scopus Eid


  • 2-s2.0-78651389634

Ro Metadata Url


  • http://ro.uow.edu.au/scipapers/3586

Number Of Pages


  • 7

Start Page


  • 326

End Page


  • 333

Volume


  • 133

Issue


  • 2

Place Of Publication


  • http://pubs.acs.org/journal/jacsat

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.

Authors


  •   Ugwumba, Isaac N. (external author)
  •   Ozawa, Kiyoshi (external author)
  •   Xu, Zhi-Qiang
  •   Ely, Fernanda (external author)
  •   Foo, Jee-Loon (external author)
  •   Herlt, Anthony J. (external author)
  •   Coppin, Chris (external author)
  •   Brown, Sue (external author)
  •   Taylor, Matthew C. (external author)
  •   Ollis, David L. (external author)
  •   Mander, Lewis N. (external author)
  •   Schenk, Gerhard (external author)
  •   Dixon, Nicholas E.
  •   Otting, Gottfried (external author)
  •   Oakeshott, John G. (external author)
  •   Jackson, Colin J. (external author)

Publication Date


  • 2011

Citation


  • Ugwumba, I. N., Ozawa, K., Xu, Z., Ely, F., Foo, J., Herlt, A. J., Coppin, C., Brown, S., Taylor, M. C., Ollis, D. L., Mander, L. N., Schenk, G., Dixon, N. E., Otting, G., Oakeshott, J. G. & Jackson, C. J. (2011). Improving a natural enzyme activity through incorporation of unnatural amino acids. Journal of the American Chemical Society, 133 (2), 326-333.

Scopus Eid


  • 2-s2.0-78651389634

Ro Metadata Url


  • http://ro.uow.edu.au/scipapers/3586

Number Of Pages


  • 7

Start Page


  • 326

End Page


  • 333

Volume


  • 133

Issue


  • 2

Place Of Publication


  • http://pubs.acs.org/journal/jacsat