Abstract
We describe here a general method for systematically replacing amino acids in an enzyme. This allows analysis of their molecular roles in substrate binding or catalysis and could eventually lead to the engineering of new enzymatic activities. The gene encoding the enzyme is first cloned into a vector from which the enzyme is expressed and is then mutated in vitro to change a particular nucleotide and hence the amino acid sequence of the enzyme. We have cloned the gene for the tyrosyl tRNA synthetase of Bacillus stearothermophilus into a vector derived from the single-stranded bacteriophage M13 to facilitate mutagenesis with mismatched synthetic oligodeoxynucleotide primers. From the recombinant M13 clone we have obtained high levels of the enzyme (∼50% of soluble protein) expressed in the Escherichia coli host and have converted cysteine (Cys35) at the enzyme's active site to serine. This leads to a reduction in enzymatic activity that is largely attributable to a lower Km for ATP.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Loftfield, R. B. Prog. Nucleic Acids Res. 12, 87–128 (1972).
Fersht, A. R. & Jakes, R. Biochemistry 14, 3350–3356 (1975).
Winter, G., Hartley, B. S., Koch, G. L. E. & Barker, D. G. (in preparation).
Barker, G. Eur. J. Biochem. 125, 357–360 (1982).
Bhat, T. N., Blow, D. M., Brick, P. & Nyborg, J. J. molec. Biol. 158, 699–709 (1982).
Monteilhet, C. & Blow, D. M. J. molec. Biol. 122, 407–417 (1978).
Rubin, J. & Blow, D. M. J. molec. Biol. 145, 489–500 (1981).
Winter, G. P., Koch, G. L. E., Dell, A. & Hartley, B. S. in Transfer RNA: Structure Properties and Recognition (eds Schimmel, P. R., Soll, D. & Abelson, J. N.) 255–265 (Cold Spring Harbor Laboratory, New York, 1979).
Barker, D. G. & Winter, G. FEBS Lett. 145, 191–193 (1982).
Zoller, M. & Smith, M. Meth. Enzym. (in the press).
Hutchison, C. A. III et al. J. biol. Chem. 253, 6551–6560 (1978).
Smith, M. & Gillam, S. in Genetic Engineering Vol. 3 (eds Setlow, J. K. & Hollaender, A.) 1 (Plenum, New York, 1981).
Wasylyk, B. et al. Proc. natn. Acad. Sci. U.S.A. 77, 7024–7028 (1980).
Miyada, C. G., Soberon, X., Itakura, K. & Wilcox, G. Gene 17, 167–177 (1982).
Gillam, S., Waterman, K. & Smith, M. Nucleic Acids Res. 2, 625–634 (1975).
Wallace, R. B., Schold, M., Johnson, M. J., Dembek, P. & Itakura, K. Nucleic Acids Res. 9, 3647–3657 (1981).
Suggs, S. V., Wallace, R. B., Hirose, T., Kawashima, E. & Itakura, K. Proc. natn. Acad. Sci. U.S.A. 78, 6613–6617 (1982).
Sanger, F., Nicklen, S. & Coulson, A. R. Proc. natn. Acad. Sci. U.S.A. 74, 5463–5467 (1977).
Clewell, D. B. & Helinski, D. R. J. Bact. 110, 1135–1146 (1972).
Hohn, B., Lechner, H. & Marvin, D. A. J. molec. Biol. 56, 143–154 (1971).
Barker, D. G., Bruton, C. J. & Winter, G. (in preparation).
McElroy, W. D., De Luca, M. & Travis, J. Science 157, 151–160 (1967).
Fersht, A. R. Biochemistry 14, 5–12 (1975).
Kerr, A. K., Ashmore, J. P. & Koetzle, T. F. Acta crystallogr. B31, 2022–2026 (1975).
Kistenmacher, T. J., Rand, G. A. & Marsh, R. E. Acta Crystallogr. B30, 2573–2578 (1974).
Crampton, M. R. in Chemistry of the −SH group (ed. Patai, S.) 379–415 (Wiley-Inter-science, New York, 1974).
Paul, I. C. in Chemistry of the −SH group (ed. Patai, S.) 111–149 (Wiley-Interecience, New York, 1974).
Fersht, A. R. & Dingwall, C. Biochemistry 18, 1245–1249 (1979).
Heinrikson, R. L. & Hartley, B. S. Biochem. J. 105, 17–24 (1967).
Sutcliffe, J. G. Cold Spring Harb. Symp. quant. Biol. 43, 77–90 (1978).
Hong, G. F. Biosci. Rep. 1, 243–252 (1981).
Messing, J. Recombinant DNA Tech. Bull. 2, 43–48 (1979).
Cohen, S. N., Chang, A. C. Y. & Hsu, L. Proc. natn. Acad. Sci. U.S.A. 69, 2110–2114 (1972).
Winter, G. & Fields, S. Nucleic Acids Res. 8, 1965–1974 (1980).
Grunstein, M. & Hogness, D. S. Proc. natn. Acad. Sci. U.S.A. 72, 3961–3965 (1975).
Denhardt, D. T. Biochem. biophys. Res. Commun. 23, 641–646 (1966).
Laemmli, U. K. Nature 227, 680–683 (1970).
Atkinson, A. et al. J. appl. Biochem. 1, 247–258 (1979).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Winter, G., Fersht, A., Wilkinson, A. et al. Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding. Nature 299, 756–758 (1982). https://doi.org/10.1038/299756a0
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/299756a0
This article is cited by
-
Current advances in design and engineering strategies of industrial enzymes
Systems Microbiology and Biomanufacturing (2021)
-
The 2018 Nobel Prize in Chemistry: Engineering proteins (enzymes/peptide/antibodies) towards desired properties via the construction of random libraries
Science China Life Sciences (2019)
-
5-Fluorouracil Co-crystals and Their Potential Anti-cancer Activities Calculated by Molecular Docking Studies
Journal of Chemical Crystallography (2016)
-
Reprogramming the genetic code
The EMBO Journal (2011)
-
Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot-Marie-Tooth neuropathy
Nature Genetics (2006)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.