Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

Tokuriki, Nobuhiko; Jackson, Colin J.; Afriat-Jurnou, Livnat; Wyganowski, Kirsten T.; Tang, Renmei; Tawfik, Dan S.

doi:10.1038/ncomms2246

Cited by 231 publications

(290 citation statements)

References 58 publications

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“…In contrast to results from in vitro evolution (18), mutations conferring diminishing returns on enzyme performance were not observed as the TrpF evolutionary trajectory proceeded. Instead, the mutation imparting the single largest improvement in TrpF activity, V15[b]M (Fig.…”

Section: Discussioncontrasting

confidence: 89%

Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

Newton

Guo

Söderholm

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

107

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New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (βα) 8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (βα) 8 barrel as an inherently evolvable protein scaffold.HisA | TrpF | adaptive evolution | enzyme performance threshold

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Section: Discussioncontrasting

confidence: 89%

Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

Newton

Guo

Söderholm

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

107

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“…2F). Large conformational changes to metal ion coordination residues and loops in the absence of metal ions are not uncommon in metallohydrolases of this superfamily (62,63).…”

Section: -Fold-increased Production Of Trznmentioning

confidence: 99%

300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

Jackson

Coppin

Carr

et al. 2014

Appl Environ Microbiol

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Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.A pproximately half of all known proteins contain a metal ion cofactor in their mature form (1). These metal ions can play structural or catalytic roles, and their incorporation into apoenzyme folding intermediates is an important step in the overall folding pathway (2). One of the most diverse superfamilies of enzymes, which includes a large number of microbial enzymes that are capable of catalyzing the hydrolysis of toxic synthetic compounds (3-5), is the metal ion-dependent amidohydrolase superfamily (6, 7). Unfortunately, many of these proteins form insoluble aggregates or are produced only at very low levels when overexpressed in recombinant systems.There is a need to improve heterologous protein expression in order to enable us to characterize proteins in the laboratory or to produce proteins on a large scale for industrial or medical purposes. Heterologous overexpression of proteins is often less than ideal because of protein aggregation or degradation (8). This can arise due to differences between the native folding situation and that which occurs when expression levels are much higher and in a different host, such as a lack of suitable folding chaperones, different pH, high local concentration of the protein, or the absence of a cofactor, which is particularly important for metalloproteins. The technique of laboratory evolution has great potential for improving soluble-protein production and involves subjecting the protein to random mutagenesis and recombination, followed by screening of the many variants that are produced for enhanced expression (9, 10)...

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“…4) (19, 22). The replacement of a carboxylated lysine in a binuclear metalloenzyme with a water molecule coordinated to the lysine upon conversion to a mononuclear enzyme has a precedent, having previously been observed in the laboratory evolution of a binuclear phosphotriesterase to a mononuclear phosphotriesterase (57). Future work could employ extended X-ray absorption fine structure or electron paramagnetic resonance analysis to define the fine details of the coordination site (58,59).…”

Section: Resultsmentioning

confidence: 91%

Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

Sugrue

Fraser

Hopkins

et al. 2015

Appl Environ Microbiol

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The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.T he amidohydrolase superfamily (AHS) has been extensively studied, with Ͼ36,000 attributed sequences since its first classification in 1998, most of which are from bacteria (1, 2). All AHS members have a (␤/␣) 8 -barrel structural fold and catalyze metaldependent hydrolysis reactions (3). The scissile bond cleaved varies between AHS enzymes, with C-O, P-O, P-S, C-N, C-S, and C-Cl bonds all having been reported to be hydrolyzed (4-8). In accordance with the metal-dependent mechanism, a mononuclear or binuclear metal binding site is observed in all AHS enzymes (9, 10). The role of the metal ion(s) in catalysis varies, depending on the substrate being hydrolyzed and the enzyme involved (10, 11). Generally, one or two metals are enlisted to lower the pK a of a catalytic water molecule, favoring the formation of a nucleophilic hydroxide (12). A second metal may polarize the substrate directly, often at a carbonyl or phosphoryl bond, or stabilize a negative charge in the transition state (13,14).Enzymes within the AHS can be separated into subtypes on the basis of the specific metal binding ligands at the conserved mononuclear or binuclear metal binding sites. Since the comprehensive review published by Seibert and Raushel in 2005 (9), in which seven subtypes were defined, two additional subtypes have been identified (Table 1) (6,15). A typical binuclear metal binding site (subtypes I, II, and VI) is characterized by a bridging ligand (generally a glutamate or carboxylated lysine residue) that coordinates both (M ␣ and M ␤ ) metal ions (16-18). Mononuclear metal binding ligands are more variable, ...

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Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

Cited by 231 publications

References 58 publications

Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

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Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

Cited by 231 publications

References 58 publications

Structural and functional innovations in the real-time evolution of new (βα) 8 barrel enzymes

Structural and functional innovations in the real-time evolution of new (βα) 8 barrel enzymes

300-Fold Increase in Production of the Zn 2+ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

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Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization