Structural Determinants of Glutathione Transferases with Azathioprine Activity Identified by DNA Shuffling of Alpha Class Members

Kurtovic, Sanela; Modén, Olof; Shokeer, Abeer; Mannervik, Bengt

doi:10.1016/j.jmb.2007.11.034

Cited by 24 publications

(15 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Up to six different GST sequences were fragmented for recombination, and in the spirit of the mythological chimeras, portions from three different organisms were combined. The resulting GSTs contained portions from human, rat, and bovine sequences (62). …”

Section: Molecular Chimerasmentioning

confidence: 99%

See 1 more Smart Citation

Five Decades with Glutathione and the GSTome

Mannervik

2012

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Uncle Folke inspired me to become a biochemist by demonstrating electrophoresis experiments on butterfly hemolymph in his kitchen. Glutathione became the subject for my undergraduate project in 1964 and has remained a focal point in my research owing to its multifarious roles in the cell. Since the 1960s, the multiple forms of glutathione transferase (GST), the GSTome, were isolated and characterized, some of which were discovered in our laboratory. Products of oxidative processes were found to be natural GST substrates. Examples of toxic compounds against which particular GSTs provide protection include 4-hydroxynonenal and ortho-quinones, with possible links to the etiology of Alzheimer and Parkinson diseases and other degenerative conditions. The role of thioltransferase and glutathione reductase in the cellular reduction of disulfides and other oxidized forms of thiols was clarified. Glyoxalase I catalyzes still another glutathione-dependent detoxication reaction. The unusual steady-state kinetics of this zinc-containing enzyme initiated model discrimination by regression analysis. Functional properties of the enzymes have been altered by stochastic mutations based on DNA shuffling and rationally tailored by structure-based redesign. We found it useful to represent promiscuous enzymes by vectors or points in multidimensional substrate-activity space and visualize them by multivariate analysis. Adopting the concept “molecular quasi-species,” we describe clusters of functionally related enzyme variants that may emerge in natural as well as directed evolution.

show abstract

Section: Molecular Chimerasmentioning

confidence: 99%

“…Ref. 62). The segments in different colors indicate the activity of a given substrate as a fraction of the sum of all the specific activities.…”

Section: Promiscuous Enzymes Represented As Vectorsmentioning

confidence: 99%

Five Decades with Glutathione and the GSTome

Mannervik

2012

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Variations in structure occur all along the sequence, as a result of the DNA shuffling as well as spurious point mutations occurring in the constructions of the library. 20 Amino acid residues distant from the active site are known to influence the catalytic efficiency of GSTs, 14,27 but substrate Fig. 10.…”

Section: Structural Features Of the Identified Quasi-speciesmentioning

confidence: 99%

“…H-site residues in Alpha class GSTs can be divided in three different regions of the primary structure: A, B, and C. Figure 15 shows hGST A1-1 in complex with an active-site ligand, Sbenzylglutathione, in which the benzyl group identifies the H-site. 20 Region A is composed of amino acids 10, 12, and 14; region B is composed of amino acids 104, 107, 108, 110, and 111; and region C, which is situated in the C-terminal helix, comprises amino acids 208, 213, 216, 220, and 222. The differences between quasi-species rA2 and hA1/A2 appear primarily in region B of the H-site (Fig.…”

Section: Structural Features Of the Identified Quasi-speciesmentioning

confidence: 99%

Emergence of Novel Enzyme Quasi-Species Depends on the Substrate Matrix

Kurtovic

Shokeer

Mannervik

2008

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

“…Protein recombination constructs libraries of hybrids by recombining fragments from two or more parents, with the goal of discovering hybrids with beneficial properties such as improved thermostability, activity, or substrate specificity [1][2][3][4][5][6][7][8][9][10][11][12][13]. For example, Stemmer demonstrated the development of beta-lactamase hybrids with a 32,000-fold increase in the required minimum inhibitory concentration of the antibiotic cefotaxime [1].…”

Section: Introductionmentioning

confidence: 99%

Protein Fragment Swapping: A Method for Asymmetric, Selective Site-Directed Recombination

Zheng

Griswold

Bailey‐Kellogg

2009

Lecture Notes in Computer Science

View full text Add to dashboard Cite

Abstract. This paper presents a new approach to site-directed recombination, swapping combinations of selected discontiguous fragments from a source protein in place of corresponding fragments of a target protein. By being both asymmetric (differentiating source and target) and selective (swapping discontiguous fragments), our method focuses experimental effort on a more restricted portion of sequence space, constructing hybrids that are more likely to have the properties that are the objective of the experiment. Furthermore, since the source and target need to be structurally homologous only locally (rather than overall), our method supports swapping fragments from functionally important regions of a source into a target "scaffold"; e.g., to humanize an exogenous therapeutic protein. A protein fragment swapping plan is defined by the residue position boundaries of the fragments to be swapped; it is assessed by an average potential score over the resulting hybrid library, with singleton and pairwise terms evaluating the importance and fit of the swapped residues. While we prove that it is NP-hard to choose an optimal set of fragments under such a potential score, we develop an integer programming approach, which we call SWAGMER, that works very well in practice. We demonstrate the effectiveness of our method in two types of swapping problem: selective recombination between beta-lactamases and activity swapping between glutathione transferases. We show that the selective recombination approach generates a better plan (in terms of resulting potential score) than a traditional site-directed recombination approach. We also show that in both cases the optimized experiment is significantly better than one that would result from stochastic methods.

show abstract

Structural Determinants of Glutathione Transferases with Azathioprine Activity Identified by DNA Shuffling of Alpha Class Members

Cited by 24 publications

References 37 publications

Five Decades with Glutathione and the GSTome

Five Decades with Glutathione and the GSTome

Emergence of Novel Enzyme Quasi-Species Depends on the Substrate Matrix

Protein Fragment Swapping: A Method for Asymmetric, Selective Site-Directed Recombination

Contact Info

Product

Resources

About