uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Transmutation of Human Glutathione Transferase A2-2 with Peroxidase Activity into an Efficient Steroid Isomerase
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
2002 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, 30019-30022 p.Article in journal (Refereed) Published
Abstract [en]

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Place, publisher, year, edition, pages
2002. Vol. 277, 30019-30022 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-89719DOI: 10.1074/jbc.M204485200PubMedID: 12023294OAI: oai:DiVA.org:uu-89719DiVA: diva2:161427
Available from: 2002-03-27 Created: 2002-03-27 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Exploring the Functional Plasticity of Human Glutathione Transferases: Allelic Variants, Novel Isoenzyme and Enzyme Redesign
Open this publication in new window or tab >>Exploring the Functional Plasticity of Human Glutathione Transferases: Allelic Variants, Novel Isoenzyme and Enzyme Redesign
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs) make up a superfamily that is involved in the cellular defense against various reactive compounds by catalyzing the conjugation of glutathione to electrophilic centra. Members of this family have also been implicated in different facets of biological signaling.

The gene encoding human GST P1-1 is polymorphic, resulting in variant amino acid residues in positions 105 and 114. The role of the polymorphism in the active-site residue 105 on enzyme stability and activity with various substrates was investigated. A valine instead of an isoleucine in position 105 decreased the thermal stability of the enzyme. The effect on enzyme activity was dependent on the substrate and reaction studied. With some substrates tested, such as carcinogenic diolepoxides derived from polyaromatic hydrocarbons, GST P1-1/Val105 displayed the highest catalytic efficiency. In contrast, with 1-chloro-2,4-dinitrobenzene, the GST P1-1/Ile105 showed higher activity. Residue 105 was mutated to alanine and tryptophan to investigate the role of size and hydrophobicity of residue 105 on enzyme properties. Generally, a smaller amino acid in position 105 gave increased activity with large substrates. Clearly, residue 105 of GST P1-1 helps to determine the substrate selectivity of the enzyme. In addition, more voluminous amino acids in position 105 increase the thermal stability of the enzyme.

GST P1-1 is believed to contribute to the development of drug resistance in cancer cells. The affinity of GST P1-1 for TER 117, designed to inhibit GST P1-1 in tumors, was not affected by the variability in position 105. TER 117 was found to be a potent inhibitor of glyoxalase I as well.

The cDNA encoding GST A3-3 was isolated from a placental cDNA library. GST A3-3 was heterologously expressed, purified and found to catalyze efficiently the double-bond isomerization of Δ5-androstene-3,17-dione and Δ5-pregnene-3,20-dione, reactions taking place in the biosynthesis of the steroid hormones testosterone and progesterone, respectively. GST A3-3 was found to be selectively expressed in steroidogenic tissues, suggesting that this enzyme is involved in the production of steroid hormones. The presence of both the hydroxyl group of the active-site tyrosine 9 and the thiolate form of glutathione, acting as a cofactor, is important for high double-bond isomerase activity. A leucine in position 111 appears to have a major role in productive binding of the steroid substrate but also residues F10 and A216 are determinants for the high isomerase activity.

GST A2-2 is a poor catalyst of the steroid double-bond isomerization of Δ5-androstene-3,17-dione as compared to GST A3-3, despite 88% sequence identity. GST A2-2 was redesigned to a highly efficient double-bond isomerase by mutating five active-site residues to the corresponding residues of GST A3-3. This demonstrates the functional plasticity of GSTs and the power of a rational approach to redesign of these enzymes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 56 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 695
Keyword
Biochemistry, Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-1858 (URN)91-554-5270-1 (ISBN)
Public defence
2002-04-19, B42, Biomedical Center, Uppsala, 10:15
Opponent
Available from: 2002-03-27 Created: 2002-03-27Bibliographically approved
2. Alpha-class glutathione transferases as steroid isomerases and scaffolds for protein redesign
Open this publication in new window or tab >>Alpha-class glutathione transferases as steroid isomerases and scaffolds for protein redesign
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The present work focuses on the glutathione transferase (GST) Alpha-class enzymes, their characteristics as steroid isomerases and structural plasticity as malleable scaffolds for protein design. The GSTs are a family of detoxication enzymes that appears to have a wider variety of additional functions.

Kinetic steady-state parameters for human GST A1-1 with the steroid isomerase substrate Δ5-androstene-3,17-dione (AD), an intermediate in steroid hormone biosynthesis, were determined. It was established that GST A1-1 is a highly efficient steroid isomerase with a 30-fold higher catalytic efficiency, in terms of kcat/Km, than 3β-hydroxysteroid dehydrogenase/Δ54-isomerase, the enzyme regarded as the mammalian Δ54-isomerase in steroid hormone biosynthesis. Kinetic parameters were also determined for GST A2-2, GST A4-4 and the GST A1-1 mutant Y9F. From the dependency on pH of the kinetic parameters it was established that efficient catalysis requires glutathione (GSH) in its deprotonated form and it is suggested that the GSH-thiolate acts as a base in the catalysis of the Δ54-3-ketosteroid isomerase reaction.

GST A2-2 is a poor catalyst of the steroid isomerase reaction while GST A3-3 is highly efficient. Their catalytic efficiencies (kcat/Km) differ 5000-fold. Stepwise point mutations were performed to GST A2-2 in order to insert the amino acid residues from the active-site of GST A3-3 that distinguishes the two isoenzymes. The result was that GST A2-2 was redesigned to a highly efficient double-bond isomerase with both the catalytic constant (kcat) and catalytic efficiency (kcat/Km) in the same order as for GST A3-3. Furthermore, this was done by only exchanging amino-acid residues with first-sphere interactions, providing empirical proof-of principle for knowledge-based enzyme design.

Kinetic studies on GST A1-1 and a T68E mutant of GST A1-1 were also performed with a GSH analog lacking the g-glutamate a-carboxylate (dGSH), and using three different electrophilic substrates (AD; 1-chloro-2,4-dinitrobenzene, CDNB; 4-nitrocinnamaldehyde). Deletion of the a-carboxylate from the GSH glutamate had a severe impact on all reaction constants and it changed the rate-limiting step for the CDNB reaction as well as changed the pKa value for the enzyme-bound GSH thiol. The loss in activity caused by dGSH could in part be compensated by the T68E mutant contributing an enzyme-bound carboxylate instead.

The C-terminus of GST A1-1 is flexible and folds over the active site when the enzyme binds a substrate. Phenylalanine residues in the C-terminal end, known to interact with active-site residues tyrosine 9 and phenylalanine 10, were mutated to abolish those interactions. Studies of viscosity dependence for CDNB and AD with regard to kcat and kcat/Km showed that the dynamic C-terminal segment influence rate-determining steps for both the larger isomerase substrate, AD, as well as for the smaller conjugation substrate, CDNB.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 38 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 719
Keyword
Biochemistry, Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-2034 (URN)91-554-5327-9 (ISBN)
Public defence
2002-05-28, BMC B22, Uppsala, 10:15
Opponent
Available from: 2002-05-03 Created: 2002-05-03 Last updated: 2014-01-27Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Authority records BETA

Mannervik, Bengt

Search in DiVA

By author/editor
Mannervik, Bengt
By organisation
Department of Biochemistry
In the same journal
Journal of Biological Chemistry
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 477 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf