Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (12)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Allen, S. J.
Right arrow Articles by Holbrook, J.J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Allen, S. J.
Right arrow Articles by Holbrook, J.J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Protein Engineering, Vol. 13, No. 1, 5-7, January 2000
© 2000 Oxford University Press

Communications

Production of an activated form of Bacillus stearothermophilus L-2-hydroxyacid dehydrogenase by directed evolution

Stuart J. Allen1 and J.John Holbrook

Molecular Recognition Centre, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk,Bristol BS8 1TD, UK

Bacillus stearothermophillus lactate dehydrogenase (bsLDH) is activated in the presence of fructose 1,6 bisphosphate (FBP). The activator is expensive and representative of the sort of co-factor complications that are undesirable in industrial processes. Three rounds of random mutagenesis and screening produced a mutant (6A) which is almost fully activated in the absence of FBP. Wild-type bsLDH has a KpyrM of 5 mM in the absence of FBP but when activated (+FBP) the KpyrM drops to 0.05 mM. The mutant 6A has a KpyrM of 0.07 mM in the absence of FBP. 6A has three amino acid substitutions—R118C, Q203L and N307S—resulting in a 70-fold activation, none of the mutations are near the active site. The activation of wild type bsLDH is due to an FBP induced tetramerization of dimeric bsLDH bringing about a structural rearrangement of key active site residues. The most likely explanation for the activation of 6A is derived from the position of Q203L, which is at the dimer–dimer interface. The suggestion is that the hydrophilic to hydrophobic change has altered the dimer–tetramer equilibrium position towards that of the tetramer. What is significant is the activation of bsLDH by a subtle long range event produced by the `blind' directed evolution approach.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Protein Eng Des SelHome page
H. Flores and A. D. Ellington
A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase
Protein Eng. Des. Sel., August 1, 2005; 18(8): 369 - 377.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.