Multi-substrate–activity space and quasi-species in enzyme evolution: Ohno's dilemma, promiscuity and functional orthogonality

2009 ◽  
Vol 37 (4) ◽  
pp. 740-744 ◽  
Author(s):  
Bengt Mannervik ◽  
Arna Runarsdottir ◽  
Sanela Kurtovic
Keyword(s):  
Multivariate Analysis ◽  
Directed Evolution ◽  
Enzyme Evolution ◽  
Activity Space ◽  
Glutathione Transferases ◽  
Multidimensional Space ◽  
Alternative Substrates ◽  
Catalytic Activities ◽  
Evolutionary Trajectories

A functional enzyme displays activity with at least one substrate and can be represented by a vector in substrate–activity space. Many enzymes, including GSTs (glutathione transferases), are promiscuous in the sense that they act on alternative substrates, and the corresponding vectors operate in multidimensional space. The direction of the vector is governed by the relative activities of the diverse substrates. Stochastic mutations of already existing enzymes generate populations of variants, and clusters of functionally similar mutants can serve as parents for subsequent generations of enzymes. The proper evolving unit is a functional quasi-species, which may not be identical with the ‘best’ variant in its generation. The manifestation of the quasi-species is dependent on the substrate matrix used to explore catalytic activities. Multivariate analysis is an approach to identifying quasi-species and to investigate evolutionary trajectories in the directed evolution of enzymes for novel functions.

scholarly journals Novel class of glutathione transferases from cyanobacteria exhibit high catalytic activities towards naturally occurring isothiocyanates

2007 ◽  
Vol 406 (1) ◽  
pp. 115-123 ◽  
Author(s):  
Eric Wiktelius ◽  
Gun Stenberg
Keyword(s):  
Catalytic Properties ◽  
Structural Features ◽  
Glutathione Transferases ◽  
Substrate Specificities ◽  
Catalytic Activities ◽  
Genome Databases ◽  
Naturally Occurring ◽  
Physiological Relevance ◽  
Associated Proteins

In the present paper, we report a novel class of GSTs (glutathione transferases), called the Chi class, originating from cyanobacteria and with properties not observed previously in prokaryotic enzymes. GSTs constitute a widespread multifunctional group of proteins, of which mammalian enzymes are the best characterized. Although GSTs have their origin in prokaryotes, few bacterial representatives have been characterized in detail, and the catalytic activities and substrate specificities observed have generally been very modest. The few well-studied bacterial GSTs have largely unknown physiological functions. Genome databases reveal that cyanobacteria have an extensive arsenal of glutathione-associated proteins. We have studied two cyanobacterial GSTs which are the first examples of bacterial enzymes that are as catalytically efficient as the best mammalian enzymes. GSTs from the thermophile Thermosynechococcus elongatus BP-1 and from Synechococcus elongatus PCC 6301 were found to catalyse the conjugation of naturally occurring plant-derived isothiocyanates to glutathione at high rates. The cyanobacterial GSTs studied are smaller than previously described members of this enzyme family, but display many of the typical structural features that are characteristics of GSTs. They are also active towards several classical substrates, but at the same moderate rates that have been observed for other GSTs derived from prokaryotes. The cloning, expression and characterization of two cyanobacterial GSTs are described. The possible significance of the observed catalytic properties is discussed in the context of physiological relevance and GST evolution.

Advances in ultrahigh-throughput screening for directed enzyme evolution

2020 ◽  
Vol 49 (1) ◽  
pp. 233-262 ◽  
Author(s):  
Ulrich Markel ◽  
Khalil D. Essani ◽  
Volkan Besirlioglu ◽  
Johannes Schiffels ◽  
Wolfgang R. Streit ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Enzyme Evolution ◽  
Screening Methods

This review summarizes how ultrahigh-throughput screening methods employ cells and biomimetic compartments to access the vast, unexplored diversity of biocatalysts with novel functions derived from directed evolution and metagenomics libraries.

scholarly journals Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

2015 ◽  
Vol 112 (47) ◽  
pp. 14599-14604 ◽  
Author(s):  
Andrew R. Buller ◽  
Sabine Brinkmann-Chen ◽  
David K. Romney ◽  
Michael Herger ◽  
Javier Murciano-Calles ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Pyrococcus Furiosus ◽  
Tryptophan Synthase ◽  
Β Subunit ◽  
Allosteric Activation ◽  
Α Subunit ◽  
X Ray ◽  
Catalytic Activities ◽  
Catalytic Potential ◽  
Protein Partners

Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.

Multidimensional epistasis and fitness landscapes in enzyme evolution

2012 ◽  
Vol 445 (1) ◽  
pp. 39-46 ◽  
Author(s):  
Wei Zhang ◽  
Daniel F. A. R. Dourado ◽  
Pedro Alexandrino Fernandes ◽  
Maria João Ramos ◽  
Bengt Mannervik
Keyword(s):  
Glutathione Transferase ◽  
Fitness Landscape ◽  
Evolutionary Process ◽  
Salient Feature ◽  
Selective Advantage ◽  
Enzyme Evolution ◽  
Conventional Analysis ◽  
Evolutionary Response ◽  
Single Protein ◽  
Evolutionary Trajectories

The conventional analysis of enzyme evolution is to regard one single salient feature as a measure of fitness, expressed in a milieu exposing the possible selective advantage at a given time and location. Given that a single protein may serve more than one function, fitness should be assessed in several dimensions. In the present study we have explored individual mutational steps leading to a triple-point-mutated human GST (glutathione transferase) A2-2 displaying enhanced activity with azathioprine. A total of eight alternative substrates were used to monitor the diverse evolutionary trajectories. The epistatic effects of the mutations on catalytic activity were variable in sign and magnitude and depended on the substrate used, showing that epistasis is a multidimensional quality. Evidently, the multidimensional fitness landscape can lead to alternative trajectories resulting in enzymes optimized for features other than the selectable markers relevant at the origin of the evolutionary process. In this manner the evolutionary response is robust and can adapt to changing environmental conditions.

scholarly journals Chemostat Approach for the Directed Evolution of Biodesulfurization Gain-of-Function Mutants

2002 ◽  
Vol 68 (2) ◽  
pp. 691-698 ◽  
Author(s):  
Joseph J. Arensdorf ◽  
A. Katrina Loomis ◽  
Philip M. DiGrazia ◽  
Daniel J. Monticello ◽  
Philip T. Pienkos
Keyword(s):  
Directed Evolution ◽  
Type Strain ◽  
Wild Type Strain ◽  
Rhodococcus Erythropolis ◽  
Organosulfur Compounds ◽  
Wild Type ◽  
Microbiological Method ◽  
Gain Of Function ◽  
Two Phase ◽  
Catalytic Activities

ABSTRACT Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies. We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of Rhodococcus erythropolis IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain. Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources. An isolate from the evolved chemostat population was genetically characterized and found to contain mutations in two genes, dszA and dszC. A transversion (G to T) in dszC codon 261 resulted in a V261F mutation that was determined to be responsible for the 5-methylbenzothiophene gain-of-function phenotype. By using a modified RACHITT (random chimeragenesis on transient templates) method, mutant DszC proteins containing all possible amino acids at that position were generated, and this mutant set was assayed for the ability to metabolize 5-methylbenzothiophene, alkyl thiophenes, and dibenzothiophene. No mutant with further improvements in these catalytic activities was identified, but several clones lost all activity, confirming the importance of codon 261 for enzyme activity.

scholarly journals Some Lactobacillusl-Lactate Dehydrogenases Exhibit Comparable Catalytic Activities for Pyruvate and Oxaloacetate

2001 ◽  
Vol 183 (1) ◽  
pp. 397-400 ◽  
Author(s):  
Kazuhito Arai ◽  
Takeo Kamata ◽  
Hiroyuki Uchikoba ◽  
Shinya Fushinobu ◽  
Hiroshi Matsuzawa ◽  
...  
Keyword(s):  
Lactobacillus Pentosus ◽  
Substrate Specificities ◽  
Reaction Velocity ◽  
Alternative Substrates ◽  
Catalytic Activities ◽  
Lactate Dehydrogenases

ABSTRACT The nonallosteric and allosteric l-lactate dehydrogenases of Lactobacillus pentosus and L. casei, respectively, exhibited broad substrate specificities, giving virtually the same maximal reaction velocity and substrateKm values for pyruvate and oxaloacetate. Replacement of Pro101 with Asn reduced the activity of the L. pentosus enzyme toward these alternative substrates to a greater extent than the activity toward pyruvate.

Directed Evolution of Protein Catalysts

2018 ◽  
Vol 87 (1) ◽  
pp. 131-157 ◽  
Author(s):  
Cathleen Zeymer ◽  
Donald Hilvert
Keyword(s):  
Directed Evolution ◽  
Rational Design ◽  
Chemical Transformations ◽  
Catalytic Promiscuity ◽  
Catalytic Activities ◽  
Effective Strategies ◽  
Wide Range ◽  
Protein Properties ◽  
New Protein ◽  
Modify Protein

Directed evolution is a powerful technique for generating tailor-made enzymes for a wide range of biocatalytic applications. Following the principles of natural evolution, iterative cycles of mutagenesis and screening or selection are applied to modify protein properties, enhance catalytic activities, or develop completely new protein catalysts for non-natural chemical transformations. This review briefly surveys the experimental methods used to generate genetic diversity and screen or select for improved enzyme variants. Emphasis is placed on a key challenge, namely how to generate novel catalytic activities that expand the scope of natural reactions. Two particularly effective strategies, exploiting catalytic promiscuity and rational design, are illustrated by representative examples of successfully evolved enzymes. Opportunities for extending these approaches to more complex biocatalytic systems are also considered.

scholarly journals Functional Diversification of Fungal Glutathione Transferases from the Ure2p Class

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Anne Thuillier ◽  
Andrew A. Ngadin ◽  
Cécile Thion ◽  
Patrick Billard ◽  
Jean-Pierre Jacquot ◽  
...  
Keyword(s):  
Extended Family ◽  
Polycyclic Aromatic Compounds ◽  
Glutathione Transferases ◽  
Transcriptional Level ◽  
Environmental Constraints ◽  
Glutathione S Transferase ◽  
Functional Diversification ◽  
Catalytic Activities ◽  
Polycyclic Aromatic ◽  
And Function

The glutathione-S-transferase (GST) proteins represent an extended family involved in detoxification processes. They are divided into various classes with high diversity in various organisms. The Ure2p class is especially expanded in saprophytic fungi compared to other fungi. This class is subdivided into two subclasses named Ure2pA and Ure2pB, which have rapidly diversified among fungal phyla. We have focused our analysis on Basidiomycetes and used Phanerochaete chrysosporium as a model to correlate the sequence diversity with the functional diversity of these glutathione transferases. The results show that among the nine isoforms found in P. chrysosporium, two belonging to Ure2pA subclass are exclusively expressed at the transcriptional level in presence of polycyclic aromatic compounds. Moreover, we have highlighted differential catalytic activities and substrate specificities between Ure2pA and Ure2pB isoforms. This diversity of sequence and function suggests that fungal Ure2p sequences have evolved rapidly in response to environmental constraints.

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