Directed evolution combined with rational design increases activity of GpdQ toward a non-physiological substrate and alters the oligomeric structure of the enzyme

Yip, Sylvia H-C., Foo, Jee-Loon, Schenk, Gerhard, Gahan, Lawrence R., Carr, Paul D. and Ollis, David L. (2011) Directed evolution combined with rational design increases activity of GpdQ toward a non-physiological substrate and alters the oligomeric structure of the enzyme. Protein Engineering Design and Selection, 24 12: 861-872. doi:10.1093/protein/gzr048


Author Yip, Sylvia H-C.
Foo, Jee-Loon
Schenk, Gerhard
Gahan, Lawrence R.
Carr, Paul D.
Ollis, David L.
Title Directed evolution combined with rational design increases activity of GpdQ toward a non-physiological substrate and alters the oligomeric structure of the enzyme
Journal name Protein Engineering Design and Selection   Check publisher's open access policy
ISSN 1741-0134
1741-0126
Publication date 2011-12
Sub-type Article (original research)
DOI 10.1093/protein/gzr048
Volume 24
Issue 12
Start page 861
End page 872
Total pages 12
Place of publication Oxford, United Kingdom
Publisher Oxford University Press
Collection year 2012
Language eng
Formatted abstract
Directed evolution was used to enhance the activity of the glycerophosphodiesterase enzyme from Enterobacter aerogenes, GpdQ, toward bis(para-nitrophenol) phosphate (BpNPP), a substrate that is frequently used to assay phosphodiesterases. Native GpdQ has a low level of activity toward BpNPP while the evolved enzymes exhibited kcat values that were well over 100 times better while improvements in kcat/Km of around 500 times were observed along with improved activity we observed a change in the oligomeric structure in the evolved enzymes. The native enzyme is a hexamer with tightly associated dimers related by a 3-fold axis. The stability of the dimer was attributed in part to the cap domain that forms a disulfide bond with its 2-fold-related subunit and in part due to the fact that dimerization results in burying 23.6% of the monomer's accessible surface area. The cap domain also forms the top of the active site and contributes an essential part of the interface between 3-fold-related molecules. The evolved proteins quickly lost one of the cysteine residues that formed the disulfide bond and other mutations that might stabilize the cap domain. The likely effect of these mutations was to open up the active site for the new substrate and to favor the formation of dimeric molecules. The breakdown of the oligomeric structure was accompanied by a reduction in the thermal stability of the protein—as monitored by the residual activity of the native and mutant proteins following pre-incubation at elevated temperatures. A discussion on the evolutionary implications of these studies is presented.
Keyword Directed evolution
Glycerophosphodiesterase
Oligomeric state
Rational design
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2012 Collection
School of Chemistry and Molecular Biosciences
 
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Created: Fri, 09 Dec 2011, 10:36:51 EST by Lucy O'Brien on behalf of School of Chemistry & Molecular Biosciences