Directed evolution of enzyme stability

Modern enzyme development relies to an increasing extent on strategies based on diversity generation followed by screening for variants with optimised properties. In principle, these directed evolution strategies might be used for optimising any enzyme property, which can be screened for in an economically feasible way, even if the molecular basis of that property is not known. Stability is an interesting property of enzymes because (1) it is of great industrial importance, (2) it is relatively easy to screen for, and (3) the molecular basis of stability relates closely to contemporary issues in protein science such as the protein folding problem and protein folding diseases. Thus, engineering enzyme stability is of both commercial and scientific interest. Here, we review how directed evolution has contributed to the development of stable enzymes and to new insight into the principles of protein stability. Several recent examples are described. These examples show that directed evolution is an effective strategy to obtain stable enzymes, especially when used in combination with rational or semi-rational engineering strategies. With respect to the principles of protein stability, some important lessons to learn from recent efforts in directed evolution are (1) that there are many structural ways to stabilize a protein, which are not always easy to rationalize, (2) that proteins may very well be stabilized by optimizing their surfaces, and (3) that high thermal stability may be obtained without forfeiture of catalytic performance at low temperatures.     

Conversion of the noncooperative Bacillus subtilis aspartate transcarbamoylase into a cooperative enzyme by a single amino acid substitution.

Allosteric enzymes are part of a unique class of enzymes which regulate metabolic pathways. On the molecular level, allosteric regulation is the result of interactions between discrete binding sites on the enzyme. In order to accommodate these multiple binding sites, allosteric enzymes have evolved with oligomeric quaternary structures. However, only a few oligomeric enzymes are known to have regulatory interactions between binding sites. Is regulatory activity an inherent property of oligomeric enzymes? The trimeric Bacillus subtilis aspartate transcarbamoylase catalyzes the first committed step of the pyrimidine biosynthetic pathway and is not known to be a regulatory enzyme. When an alanine residue is substituted for the active-site residue Arg-99 by site-specific mutagenesis, the regulatory activity of homotropic substrate cooperativity (Hill coefficient of 1.5) is observed in the resulting mutant enzyme. These results suggest that homotropic regulation may have evolved by a relatively small number of mutations to an oligomeric enzyme.     

Differences in enzymatic properties allow SodCI but not SodCII to contribute to virulence in Salmonella enterica serovar Typhimurium strain 14028

Salmonella enterica serovar Typhimurium produces two Cu/Zn cofactored periplasmic superoxide dismutases, SodCI and SodCII. While mutations in sodCI attenuate virulence eightfold, loss of SodCII does not confer a virulence phenotype, nor does it enhance the defect observed in a sodCI background. Despite this in vivo phenotype, SodCI and SodCII are expressed at similar levels in vitro during the stationary phase of growth. By exchanging the open reading frames of sodCI and sodCII, we found that SodCI contributes to virulence when placed under the control of the sodCII promoter. In contrast, SodCII does not contribute to virulence even when expressed from the sodCI promoter. Thus, the disparity in virulence phenotypes is due primarily to some physical difference between the two enzymes. In an attempt to identify the unique property of SodCI, we have tested factors that might affect enzyme activity inside a phagosome. We found no significant difference between SodCI and SodCII in their resistance to acid, resistance to hydrogen peroxide, or ability to obtain copper in a copper-limiting environment. Both enzymes are synthesized as apoenzymes in the absence of copper and can be fully remetallated when copper is added. The one striking difference that we noted is that, whereas SodCII is released normally by an osmotic shock, SodCI is "tethered" within the periplasm by an apparently noncovalent interaction. We propose that this novel property of SodCI is crucial to its ability to contribute to virulence in serovar Typhimurium.     

The role of log-normal dynamics in the evolution of biochemical pathways

The study of the scale-free topology in non-biological and biological networks and the dynamics that can explain this fascinating property of complex systems have captured the attention of the scientific community in the last years. Here, we analyze the biochemical pathways of three organisms (Methanococcus jannaschii, Escherichia coli, Saccharomyces cerevisiae) which are representatives of the main kingdoms Archaea, Bacteria and Eukaryotes during the course of the biological evolution. We can consider two complementary representations of the biochemical pathways: the enzymes network and the chemical compounds network. In this article, we propose a stochastic model that explains that the scale-free topology with exponent in the vicinity of gamma approximately 3/2 found across these three organisms is governed by the log-normal dynamics in the evolution of the enzymes network. Precisely, the fluctuations of the connectivity degree of enzymes in the biochemical pathways between evolutionary distant organisms follow the same conserved dynamical principle, which in the end is the origin of the stationary scale-free distribution observed among species, from Archaea to Eukaryotes. In particular, the log-normal dynamics guarantees the conservation of the scale-free distribution in evolving networks. Furthermore, the log-normal dynamics also gives a possible explanation for the restricted range of observed exponents gamma in the scale-free networks (i.e., gamma > or = 3/2). Finally, our model is also applied to the chemical compounds network of biochemical pathways and the Internet network.     

Evaluating limited specificity of drug pumps reduced relative resistance in human MDR phenotypes.

In the parallel paper, we developed a property to characterize drug efflux pumps, i.e. the reduced relative resistance (RRR). Using this RRR, we here investigate whether the observed diversity in human multidrug resistance (MDR) phenotypes might be due to variable levels of P-glycoprotein encoded by MDR1. We analyzed resistance phenotypes of various human cell lines in which either one, or both, classical human multidrug resistance genes, MDR1 and MDR3, are overexpressed. In addition, RRR values were calculated for MDR phenotypes presented in the literature. The results suggest that more than a single mechanism is required to account for the observed phenotypic diversity of classical multidrug resistance. This diversity is only partly due to differences in plasma membrane permeabilities between cell line families. It is discussed whether the alternative MDR phenotypes might be MDR1 phenotypes modified by other factors that do not themselves cause MDR. The method we here apply may also be useful for other nonspecific enzymes or pumps.     

Conformational Variation in Enzyme Catalysis: A Structural Study on Catalytic Residues

Conformational variation in catalytic residues can be captured as alternative snapshots in enzyme crystal structures. Addressing the question of whether active site flexibility is an intrinsic and essential property of enzymes for catalysis, we present a comprehensive study on the 3D variation of active sites of 925 enzyme families, using explicit catalytic residue annotations from the Mechanism and Catalytic Site Atlas and structural data from the Protein Data Bank. Through weighted pairwise superposition of the functional atoms of active sites, we captured structural variability at single-residue level and examined the geometrical changes as ligands bind or as mutations occur. We demonstrate that catalytic centres of enzymes can be inherently rigid or flexible to various degrees according to the function they perform, and structural variability most often involves a subset of the catalytic residues, usually those not directly involved in the formation or cleavage of bonds. Moreover, data suggest that 2/3 of active sites are flexible, and in half of those, flexibility is only observed in the side chain. The goal of this work is to characterise our current knowledge of the extent of flexibility at the heart of catalysis and ultimately place our findings in the context of the evolution of catalysis as enzymes evolve new functions and bind different substrates.     

Troglitazone inhibits expression of the phosphoenolpyruvate carboxykinase gene by an insulin-independent mechanism.

Troglitazone is an oral insulin-sensitizing drug used to treat patients with type 2 diabetes. A major feature of this hyperglycemic state is the presence of increased rates of hepatic gluconeogenesis, which troglitazone is able to ameliorate. In this study, we examined the molecular basis for this property of troglitazone by exploring the effects of this compound on the expression of the two genes encoding the major regulatory enzymes of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary cultures of rat hepatocytes. Insulin is able to inhibit expression of both of these genes, which was verified in our model system. Troglitazone significantly reduced mRNA levels of PEPCK and G6Pase in rat hepatocytes isolated from normal and Zucker-diabetic rats, but to a lesser extent than that observed with insulin. Interestingly, troglitazone was unable to reduce cAMP-induced levels of PEPCK mRNA, suggesting that the molecular mechanism whereby troglitazone exerted its effects on gene expression differed from that of insulin. This was further supported by the observation that troglitazone was able to reduce PEPCK mRNA levels in the presence of the insulin signaling pathway inhibitors wortmannin, rapamycin, and PD98059. These results indicate that troglitazone can regulate the expression of specific genes in an insulin-independent manner, and that genes encoding gluconeogenic enzymes are targets for the inhibitory effects of this drug.     

Interaction of tetrahydrofolate and other folate derivatives with bacterial sarcosine oxidase

Sarcosine oxidase from Corynebacterium sp. P-1 binds 2 mol of tetrahydrofolate/mol of enzyme (KD = 8.8 microM). The same stoichiometry is observed with tetrahydropteroyltetraglutamate (KD = 15.4 microM). Binding is also observed with pteroyltetraglutamate and with 5-formyltetrahydrofolate. In the case of the pteroylmonoglutamates, binding appears to be sensitive to changes in the pteridine ring since no binding is observed with 5-methyltetrahydrofolate or with folate. Sarcosine oxidase can be specifically adsorbed onto an affinity matrix prepared by coupling 5-formyltetrahydrofolate to AH-Sepharose. Tetrahydrofolate does not affect the rate of sarcosine oxidation but does block the formation of formaldehyde as a final product. In the presence of tetrahydrofolate, sarcosine oxidation is accompanied by the formation of 5,10-methylenetetrahydrofolate at a rate that exceeds the rate at which formaldehyde (or a precursor) can be released into solution and which is also considerably faster than the nonenzymic reaction of free formaldehyde with tetrahydrofolate. It is suggested that tetrahydrofolate may serve primarily to trap formaldehyde as it is formed at the active site during sarcosine oxidation. The existence of a catalytically significant binding site for tetrahydrofolate appears to be a general property of sarcosine oxidizing enzymes since similar results have previously been obtained with mammalian sarcosine dehydrogenase, an enzyme that is structurally and mechanistically very different from bacterial sarcosine oxidase.     

Global self-organization of the cellular metabolic structure

Background

Over many years, it has been assumed that enzymes work either in an isolated way, or organized in small catalytic groups. Several studies performed using “metabolic networks models” are helping to understand the degree of functional complexity that characterizes enzymatic dynamic systems. In a previous work, we used “dissipative metabolic networks” (DMNs) to show that enzymes can present a self-organized global functional structure, in which several sets of enzymes are always in an active state, whereas the rest of molecular catalytic sets exhibit dynamics of on-off changing states. We suggested that this kind of global metabolic dynamics might be a genuine and universal functional configuration of the cellular metabolic structure, common to all living cells. Later, a different group has shown experimentally that this kind of functional structure does, indeed, exist in several microorganisms.

Methodology/Principal Findings

Here we have analyzed around 2.500.000 different DMNs in order to investigate the underlying mechanism of this dynamic global configuration. The numerical analyses that we have performed show that this global configuration is an emergent property inherent to the cellular metabolic dynamics. Concretely, we have found that the existence of a high number of enzymatic subsystems belonging to the DMNs is the fundamental element for the spontaneous emergence of a functional reactive structure characterized by a metabolic core formed by several sets of enzymes always in an active state.

Conclusions/Significance

This self-organized dynamic structure seems to be an intrinsic characteristic of metabolism, common to all living cellular organisms. To better understand cellular functionality, it will be crucial to structurally characterize these enzymatic self-organized global structures.     

Mathematical modelling of the purine metabolism of the rat liver

The regulation of the purine metabolism of the rat liver is studied on the basis of a mathematical model which comprises rate laws and kinetic constants of all physiologically relevant reactions. The computed stationary and time-dependent concentrations are in good accordance with experimental data obtained in the ischaemic rat liver and in isolated hepatocytes. In particular, model-based simulations of the adenine nucleotide metabolism have been performed for situations where ATP-deficient states of the cell (hypoxia, anoxia or ischaemia) of various length are followed by onset of ATP production (reoxygenation). These simulations confirm the experimentally observed incomplete recovery of ATP and of the total pool of adenine nucleotides within a few hours of reoxygenation after long-term ATP depletion. Therefore, it can be concluded that this phenomenon is an intrinsic regulatory property of the purine metabolism and not necessarily due to some irreversible changes in the activity of the enzymes involved.     

The distribution of restriction enzyme sites in Escherichia coli.

A statistical analysis of physical map data for eight restriction enzymes covering nearly the entire genome of E. coli is presented. The methods of analysis are based on a top-down modeling approach which requires no knowledge of the statistical properties of the base sequence. For most enzymes, the distribution of mapped sites is found to be fairly homogeneous. Some heterogeneity in the distribution of sites is observed for the enzymes Pstl and HindIII. In addition, BamHI sites are found to be more evenly dispersed than we would expect for random placement and we speculate on a possible mechanism. A consistent departure from a uniform distribution, observed for each of the eight enzymes, is found to be due to a lack of closely spaced sites. We conclude from our analysis that this departure can be accounted for by deficiencies in the physical map data rather than non-random placement of actual restriction sites. Estimates of the numbers of sites missing from the map are given, based both on the map data itself and on the site frequencies in a sample of sequenced E. coli DNA. We conclude that 5 to 15% of the mapped sites represent multiple sites in the DNA sequence.     

Regulation of enzymes of the 3,5-xylenol-degradative pathway in Pseudomonas putida: evidence for a plasmid.

Constitutive synthesis of enzymes responsible for methyl group oxidation in 3,5-xylenol degradation and an associated p-cresol methylhydroxylase in Pseudomonas putida NCIB 9869 was shown by their retention at high specific activities in cells transferred from 3,5-xylenol medium to glutamate medium. The specific activities of other enzymes of the 3,5-xylenol pathway declined upon removal of aromatic substrate, consistent with their inducible control. Specific activities of the methyl-oxidizing enzymes showed an eventual decline concomitant with a decrease in the fraction of bacteria capable of growth with 3,5-xylenol; a simultaneous loss of the ability to grow with m-hydroxybenzoate was also observed. The property of 3,5-xylenol utilization could be transferred to another strain of P. putida. It is proposed that enzymes of the 3,5-xylenol pathway and those for conversion of p-cresol to p-hydroxybenzoate are plasmid encoded, that the early methyl-oxidizing enzymes are expressed constitutively, and that the later enzymes are inducible.     

Bacterial gene regulation in diauxic and non-diauxic growth

When bacteria are grown in a batch culture containing a mixture of two growth-limiting substrates, they exhibit a rich spectrum of substrate consumption patterns including diauxic growth, simultaneous consumption, and bistable growth. In previous work, we showed that a minimal model accounting only for enzyme induction and dilution captures all the substrate consumption patterns [Narang, A., 1998a. The dynamical analogy between microbial growth on mixtures of substrates and population growth of competing species. Biotechnol. Bioeng. 59, 116-121, Narang, A., 2006. Comparitive analysis of some models of gene regulation in mixed-substrate microbial growth, J. Theor. Biol. 242, 489-501]. In this work, we construct the bifurcation diagram of the minimal model, which shows the substrate consumption pattern at any given set of parameter values. The bifurcation diagram explains several general properties of mixed-substrate growth. (1) In almost all the cases of diauxic growth, the "preferred" substrate is the one that, by itself, supports a higher specific growth rate. In the literature, this property is often attributed to the optimality of regulatory mechanisms. Here, we show that the minimal model, which accounts for induction and growth only, displays the property under fairly general conditions. This suggests that the higher growth rate of the preferred substrate is an intrinsic property of the induction and dilution kinetics. It can be explained mechanistically without appealing to optimality principles. (2) The model explains the phenotypes of various mutants containing lesions in the regions encoding for the operator, repressor, and peripheral enzymes. A particularly striking phenotype is the "reversal of the diauxie" in which the wild-type and mutant strains consume the very same two substrates in opposite order. This phenotype is difficult to explain in terms of molecular mechanisms, such as inducer exclusion or CAP activation, but it turns out to be a natural consequence of the model. We show furthermore that the model is robust. The key property of the model, namely, the competitive dynamics of the enzymes, is preserved even if the model is modified to account for various regulatory mechanisms. Finally, the model has important implications for the problem of size regulation in development. It suggests that protein dilution may be the mechanism coupling patterning and growth.     

Stability of positional identity of axolotl blastema cells in vitro

Summary Previous grafting experiments have demonstrated that cells from non-contiguous positions within developing and regenerating limbs differ in a property referred to as positional identity. The goal of this study was to determine how long the positional identity of axolotl limb blastema cells is stable during culture in vitro. We have developed an assay for posterior positional properties such that blastema cells can be cultured and then grafted into anterior positions in host blastemas, to determine if they can stimulate supernumerary digit formation. We report that posterior blastema cells are able to maintain their positional identities for at least a week in culture. In addition, we observed that blastema cells are able to rapidly degrade collagenous substrates in vitro, a property that apparently distinguishes them from limb cells of other vertebrates. These results provide information regarding the time boundaries within which the positional properties of blastema cells can be studied and manipulated in vitro. Correspondence to: S.V. Bryant     

G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin''s peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.     

A C4-oxidizing Lytic Polysaccharide Monooxygenase Cleaving Both Cellulose and Cello-oligosaccharides

Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61–3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end.     

The inflow of the substrate can regulate the persistence of memory of enzymes having the properties of hysteresis: theoretical analysis

A simple kinetic model of hysteretic enzymes with the influx of substrate or ion (transported by the enzyme) is considered. Two alternative steady activity levels are shown to arise in the system with a hysteretic enzyme. The transition between these levels can proceed in an oscillatory manner. The duration of the initial steady activity level is shown to be determined by the initial substrate (or ion) level, and the oscillatory transition between the activity levels is the property of hysteretic enzymes. It was shown for plasma membrane Ca2+-ATPase as an example that the level of the signal can be encoded into the time interval in which the enzyme retains the memory about this signal.     

Fluxes and metabolic pools as model traits for quantitative genetics. I. The L-shaped distribution of gene effects

The fluxes through metabolic pathways can be considered as model quantitative traits, whose QTL are the polymorphic loci controlling the activity or quantity of the enzymes. Relying on metabolic control theory, we investigated the relationships between the variations of enzyme activity along metabolic pathways and the variations of the flux in a population with biallelic QTL. Two kinds of variations were taken into account, the variation of the average enzyme activity across the loci, and the variation of the activity of each enzyme of the pathway among the individuals of the population. We proposed analytical approximations for the flux mean and variance in the population as well as for the additive and dominance variances of the individual QTL. Monte Carlo simulations based on these approximations showed that an L-shaped distribution of the contributions of individual QTL to the flux variance (R(2)) is consistently expected in an F(2) progeny. This result could partly account for the classically observed L-shaped distribution of QTL effects for quantitative traits. The high correlation we found between R(2) value and flux control coefficients variance suggests that such a distribution is an intrinsic property of metabolic pathways due to the summation property of control coefficients.     

Vibrio cholerae periplasmic superoxide dismutase: isolation of the gene and overexpression of the protein

Superoxide dismutases are ubiquitous enzymes which play an important role in protecting cells against oxidative damage and which have also been shown to contribute to the pathogenicity of many bacterial species. Here we demonstrate that Vibrio cholerae, the causative agent of cholerae, expresses an active periplasmic Cu,Zn superoxide dismutase. Moreover, we have set up an expression system yielding large amounts of V. cholerae recombinant Cu,Zn superoxide dismutase in the periplasm of Escherichia coli and a procedure to obtain the enzyme in a highly purified form. Unlike the bovine enzyme, V. cholerae Cu,Zn superoxide dismutase has been proved to be highly resistant to inactivation by hydrogen peroxide. This property, which appears to be common to other bacterial enzymes of this class, might improve the ability of Cu,Zn superoxide dismutase to protect bacteria against the reactive oxygen species produced by phagocytes.