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Engineering GST M2-2 for High Activity with Indene 1,2-Oxide and Indication of an H-SiteResidue Sustaining Catalytic Promiscuity
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (biokemi och organisk kemi)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (biokemi och organisk kemi)
2011 (English)In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 412, no 1, 111-120 p.Article in journal (Refereed) Published
Abstract [en]

The substrate-binding H-site of human glutathionetransferase (GST) M2-2 was subjected to iterative saturation mutagenesis in order to obtain an efficient enzyme with the novel epoxide substrate indene 1,2-oxide. Residues 10, 116, and 210 were targeted, and the activities with the alternative substrates, benzyl isothiocyanate and the prodrug azathioprine, undergoing divergent chemical reactions were monitored for comparison. In general, increased activities were found when the smaller residues Gly, Ser, and Ala replaced the original Thr210. The most active mutant T210G was further mutated at position 116, but no mutant showed enhanced catalytic activity. However, saturation mutagenesis of position 10 identified one double mutant T210G/I10C with 100-fold higher specific activity with indene 1,2-oxide than wild-type GST M2-2. This enhanced epoxide activity of 50 mu mol min(-1) mg(-1) resulted primarily from an increased k(cat) value (70 s(-1)). The specific activity is 24-fold higher than that of wild-type GST M1-1, which is otherwise the most proficient GST enzyme with epoxide substrates. A second double mutant T210G/I10W displayed 30-fold increased activity with azathioprine, 0.56 mu mol min(-1) mg(-1). In both double mutants, the replacement of Ile10 led to narrowed acceptance of alternative substrates. Ile10 is evolutionarily conserved in related class Mu GSTs. Conservation usually indicates preservation of a particular function, and in the Mu class, it would appear that the conservedIle10 is not necessary to maintain catalytic functions but to prevent loss of broad substrate acceptance. In summary, our data underscore the facile transition between alternative substrateselectivity profiles in GSTs by a few mutations. 

Place, publisher, year, edition, pages
2011. Vol. 412, no 1, 111-120 p.
Keyword [en]
protein redesign, active site, conserved residue, saturation mutagenesis, iterative saturation mutagenesis, glutathione transferase, GST M2-2, substrate selectivity, promiscuous
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-149325DOI: 10.1016/j.jmb.2011.07.039ISI: 000294523300011OAI: oai:DiVA.org:uu-149325DiVA: diva2:404546
Available from: 2011-03-17 Created: 2011-03-17 Last updated: 2013-06-20Bibliographically approved
In thesis
1. Modulating Enzyme Functions by Semi-Rational Redesign and Chemical Modifications: A Study on Mu-class Glutathione Transferases
Open this publication in new window or tab >>Modulating Enzyme Functions by Semi-Rational Redesign and Chemical Modifications: A Study on Mu-class Glutathione Transferases
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Today, enzymes are extensively used for many industrial applications, this includes bulk and fine-chemical synthesis, pharmaceuticals and consumer products. Though Nature has perfected enzymes for many millions of years, they seldom reach industrial performance targets. Natural enzymes could benefit from protein redesign experiments to gain novel functions or optimize existing functions.

Glutathione transferases (GSTs) are detoxification enzymes, they also display disparate functions. Two Mu-class GSTs, M1-1 and M2-2, are closely related but display dissimilar substrate selectivity profiles. Saturation mutagenesis of a previously recognized hypervariable amino acid in GST M2-2, generated twenty enzyme variants with altered substrate selectivity profiles, as well as modified thermostabilities and expressivities. This indicates an evolutionary significance; GST Mu-class enzymes could easily alter functions in a duplicate gene by a single-point mutation.

To further identify residues responsible for substrate selectivity in the GST M2-2 active site, three residues were chosen for iterative saturation mutagenesis. Mutations in position10, identified as highly conserved, rendered enzyme variants with substrate selectivity profiles resembling that of specialist enzymes. Ile10 could be conserved to sustain the broad substrate acceptance displayed by GST Mu-class enzymes.

Enzymes are constructed from primarily twenty amino acids, it is a reasonable assumption that expansion of the amino acid repertoire could result in functional properties that cannot be accomplished with the natural set of building blocks. A combination approach of site-directed mutagenesis and chemical modifications in GST M2-2 and GST M1-1 resulted in novel enzyme variants that displayed altered substrate selectivity patterns as well as improved enantioselectivities.

The results presented in this thesis demonstrate the use of different protein redesign techniques to modulate various functions in Mu-class GSTs. These techniques could be useful in search of optimized enzyme variants for industrial targets.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 741
Keyword
protein redesign, semi-rational redesign, saturation mutagenesis, iterative saturation mutagenesis, chemical modification, Cys, Cys-X scanning, enzyme evolution, promiscuous, substrate selectivity, enantioselectivity
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-149326 (URN)978-91-554-8029-5 (ISBN)
Public defence
2011-04-29, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
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Supervisors
Note
biokemi och organisk kemiAvailable from: 2011-04-08 Created: 2011-03-17 Last updated: 2011-05-05Bibliographically approved

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