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Modulating Enzyme Functions by Semi-Rational Redesign and Chemical Modifications: A Study on Mu-class Glutathione Transferases
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
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 [en]
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: urn:nbn:se:uu:diva-149326ISBN: 978-91-554-8029-5OAI: oai:DiVA.org:uu-149326DiVA: diva2:404564
Public defence
2011-04-29, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Note
biokemi och organisk kemiAvailable from: 2011-04-08 Created: 2011-03-17 Last updated: 2011-05-05Bibliographically approved
List of papers
1. Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution
Open this publication in new window or tab >>Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution
2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 13, 4876-4881 p.Article in journal (Refereed) Published
Abstract [en]

All molecular species in an organism are connected physically and functionally to other molecules. In evolving systems, it is not obvious to what extent functional properties of a protein can change to selective advantage and leave intact favorable traits previously acquired. This uncertainty has particular significance in the evolution of novel pathways for detoxication, because an organism challenged with new xenobiotics in the environment may still require biotransformation of previously encountered toxins. Positive selection has been proposed as an evolutionary mechanism for facile adaptive responses of proteins to changing conditions. Here, we show, by saturation mutagenesis, that mutations of a hypervariable residue in human glutathione transferase M2-2 can differentially change the enzyme's substrate-activity profile with alternative substrates and, furthermore, enable or disable dissimilar chemical reactions. Crystal structures demonstrate that activity with epoxides is enabled through removal of steric hindrance from a methyl group, whereas activities with an orthoquinone and a nitroso donor are maintained in the variant enzymes. Given the diversity of cellular activities in which a single protein can be engaged, the selective transmutation of functional properties has general significance in molecular evolution.

Keyword
Enzyme Stability, Epoxy Compounds/metabolism, Evolution; Molecular, Glutathione Transferase/chemistry/*genetics/*metabolism, Humans, Models; Molecular, Molecular Sequence Data, Molecular Structure, Mutation/genetics, Protein Structure; Tertiary, Research Support; Non-U.S. Gov't, Serine/chemistry/metabolism, Substrate Specificity, Temperature, Threonine/chemistry/metabolism, Variation (Genetics)/genetics
Identifiers
urn:nbn:se:uu:diva-81516 (URN)10.1073/pnas.0600849103 (DOI)16549767 (PubMedID)
Available from: 2008-01-29 Created: 2008-01-29 Last updated: 2013-07-18Bibliographically approved
2. Engineering GST M2-2 for High Activity with Indene 1,2-Oxide and Indication of an H-SiteResidue Sustaining Catalytic Promiscuity
Open this publication in new window or tab >>Engineering GST M2-2 for High Activity with Indene 1,2-Oxide and Indication of an H-SiteResidue Sustaining Catalytic Promiscuity
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. 

Keyword
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:nbn:se:uu:diva-149325 (URN)10.1016/j.jmb.2011.07.039 (DOI)000294523300011 ()
Available from: 2011-03-17 Created: 2011-03-17 Last updated: 2013-06-20Bibliographically approved
3. Cys-X Scanning for Expansion of Active-site Residues and Modulation of Catalytic Functions in a Glutathione Transferase
Open this publication in new window or tab >>Cys-X Scanning for Expansion of Active-site Residues and Modulation of Catalytic Functions in a Glutathione Transferase
2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 19, 16871-16878 p.Article in journal (Refereed) Published
Abstract [en]

We propose Cys-X scanning as a semisynthetic approach to engineer the functional properties of recombinant proteins. As in the case of Ala scanning, key residues in the primary structure are identified, and one of them is replaced by Cys via site-directed mutagenesis. The thiol of the residue introduced is subsequently modified by alternative chemical reagents to yield diverse Cys-X mutants of the protein. This chemical approach is orthogonal to Ala or Cys scanning and allows the expansion of the repertoire of amino acid side chains far beyond those present in natural proteins. In its present application, we have introduced Cys-X residues in human glutathione transferase (GST) M2-2, replacing Met-212 in the substrate-binding site. To achieve selectivity of the modifications, the Cys residues in the wild-type enzyme were replaced by Ala. A suite of simple substitutions resulted in a set of homologous Met derivatives ranging from normethionine to S-heptyl-cysteine. The chemical modifications were validated by HPLC and mass spectrometry. The derivatized mutant enzymes were assayed with alternative GST substrates representing diverse chemical reactions: aromatic substitution, epoxide opening, transnitrosylation, and addition to an ortho-quinone. The Cys substitutions had different effects on the alternative substrates and differentially enhanced or suppressed catalytic activities depending on both the Cys-X substitution and the substrate assayed. As a consequence, the enzyme specificity profile could be changed among the alternative substrates. The procedure lends itself to large-scale production of Cys-X modified protein variants.

Place, publisher, year, edition, pages
The American Society for Biochemistry and Molecular Biology, Inc., 2011
Keyword
protein redesign, chemical modification, substrate selectivity, Cys, enzyme evolution, glutathione transferases, GST M2-2, Cys-X scanning
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-149319 (URN)10.1074/jbc.M111.230078 (DOI)000290301900036 ()21454564 (PubMedID)
Available from: 2011-03-17 Created: 2011-03-17 Last updated: 2011-11-10Bibliographically approved
4. Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications
Open this publication in new window or tab >>Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications
2007 (English)In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1770, no 9, 1374-1381 p.Article in journal (Refereed) Published
Abstract [en]

Based on the crystal structure of human glutathione transferase M1-1, cysteine residues were introduced in the substrate-binding site of a Cys-free mutant of the enzyme, which were subsequently alkylated with 1-iodoalkanes. By different combinations of site-specific mutations and chemical modifications of the enzyme the enantioselectivity in the conjugation of glutathione with the epoxide-containing substrates 1-phenylpropylene oxide and styrene-7,8-oxide were enhanced up to 9- and 10-fold. The results also demonstrate that the enantioselectivity can be diminished, or even reversed, by suitable modifications, which can be valuable under some conditions. The redesign of the active-site structure for enhanced or diminished enantioselectivities have divergent requirements for different epoxides, calling for a combinatorial approach involving alternative mutations and chemical modifications to optimize the enantioselectivity for a targeted substrate. This approach outlines a general method of great potential for fine-tuning substrate specificity and tailoring stereoselectivity of recombinant enzymes.

Keyword
Enantioselectivity, Epoxide resolution, Glutathione transferase, Protein modification, Rational redesign
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-11976 (URN)10.1016/j.bbagen.2007.06.002 (DOI)000249511200014 ()17689871 (PubMedID)
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2013-07-09Bibliographically approved

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