mRNA Display Pipeline for Protein Biosensor Construction.

Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >103 unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties.

Generalizable Protein Biosensors Based on Synthetic Switch Modules.

Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor's activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.

Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its ß site in the absence of a substrate. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and in the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the ß site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the ß site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first- and second-coordination sphere residues at the ß site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second-sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.

Ca(II) Binding Regulates and Dominates the Reactivity of a Transition-Metal-Ion-Dependent Diesterase from Mycobacterium tuberculosis.

The diesterase Rv0805 from Mycobacterium tuberculosis is a dinuclear metallohydrolase that plays an important role in signal transduction by controlling the intracellular levels of cyclic nucleotides. As Rv0805 is essential for mycobacterial growth it is a promising new target for the development of chemotherapeutics to treat tuberculosis. The in vivo metal-ion composition of Rv0805 is subject to debate. Here, we demonstrate that the active site accommodates two divalent transition metal ions with binding affinities ranging from approximately 50 nm for Mn(II) to about 600 nm for Zn(II) . In contrast, the enzyme GpdQ from Enterobacter aerogenes, despite having a coordination sphere identical to that of Rv0805, binds only one metal ion in the absence of substrate, thus demonstrating the significance of the outer sphere to modulate metal-ion binding and enzymatic reactivity. Ca(II) also binds tightly to Rv0805 (Kd ≈40 nm), but kinetic, calorimetric, and spectroscopic data indicate that two Ca(II) ions bind at a site different from the dinuclear transition-metal-ion binding site. Ca(II) acts as an activator of the enzymatic activity but is able to promote the hydrolysis of substrates even in the absence of transition-metal ions, thus providing an effective strategy for the regulation of the enzymatic activity.

Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH).

Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH.

Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry.

Binuclear metallohydrolases are a large and diverse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k cat, K m, and k cat/K m). Continuous spectroscopic assays are frequently only possible by using synthetic (i.e., nonbiological) substrates that possess a suitable chromophoric marker (e.g., nitrophenol). Isothermal titration calorimetry, in contrast, affords a rapid assay independent of the chromophoric properties of the substrate-the heat associated with the hydrolytic reaction can be directly related to catalytic properties. Here, we demonstrate the efficiency of the method on several selected examples of this family of enzymes and show that, in general, the catalytic parameters obtained by isothermal titration calorimetry are in good agreement with those obtained from spectroscopic assays.

Identification and characterization of an unusual metallo-ß-lactamase from Serratia proteamaculans.

Metallo-ß-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing ß-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK a values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn(2+) center is formed only once the substrate is present.

Phosphate-bound structure of an organophosphate-degrading enzyme from Agrobacterium radiobacter.

OpdA is a binuclear metalloenzyme that can hydrolyze organophosphate pesticides and nerve agents. In this study the crystal structure of the complex between OpdA and phosphate has been determined to 2.20 Å resolution. The structure shows the phosphate bound in a tripodal mode to the metal ions whereby two of the oxygen atoms of PO(4) are terminally bound to each metal ion and a third oxygen bridges the two metal ions, thus displacing the µOH in the active site. In silico modelling demonstrates that the phosphate moiety of a reaction product, e.g. diethyl phosphate, may bind in the same orientation, positioning the diethyl groups neatly into the substrate binding pocket close to the metal center. Thus, similar to the binuclear metallohydrolases urease and purple acid phosphatase the tripodal arrangement of PO(4) is interpreted in terms of a role of the µOH as a reaction nucleophile.

Electronic and geometric structures of the organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA).

The organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA) is a highly efficient catalyst for the degradation of pesticides and some nerve agents such as sarin. OpdA requires two metal ions for catalytic activity, and hydrolysis is initiated by a nucleophilic hydroxide that is bound to one of these metal ions. The precise location of this nucleophile has been contentious, with both a terminal and a metal-ion-bridging hydroxide as likely candidates. Here, we employed magnetic circular dichroism to probe the electronic and geometric structures of the Co(II)-reconstituted dinuclear metal center in OpdA. In the resting state the metal ion in the more secluded α site is five-coordinate, whereas the Co(II) in the solvent-exposed ß site is predominantly six-coordinate with two terminal water ligands. Addition of the slow substrate diethyl 4-methoxyphenyl phosphate does not affect the α site greatly but lowers the coordination number of the ß site to five. A reduction in the exchange coupling constant indicates that substrate binding also triggers a shift of the µ-hydroxide into a pseudoterminal position in the coordination sphere of either the α or the ß metal ion. Mechanistic implications of these observations are discussed.

Improving a natural enzyme activity through incorporation of unnatural amino acids.

The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

The organophosphate-degrading enzyme from Agrobacterium radiobacter displays mechanistic flexibility for catalysis.

The OP (organophosphate)-degrading enzyme from Agrobacterium radiobacter (OpdA) is a binuclear metallohydrolase able to degrade highly toxic OP pesticides and nerve agents into less or non-toxic compounds. In the present study, the effect of metal ion substitutions and site-directed mutations on the catalytic properties of OpdA are investigated. The study shows the importance of both the metal ion composition and a hydrogen-bond network that connects the metal ion centre with the substrate-binding pocket using residues Arg254 and Tyr257 in the mechanism and substrate specificity of this enzyme. For the Co(II) derivative of OpdA two protonation equilibria (pKa1 ~5; pKa2 ~10) have been identified as relevant for catalysis, and a terminal hydroxide acts as the likely hydrolysis-initiating nucleophile. In contrast, the Zn(II) and Cd(II) derivatives only have one relevant protonation equilibrium (pKa ~4-5), and the µOH is the proposed nucleophile. The observed mechanistic flexibility may reconcile contrasting reaction models that have been published previously and may be beneficial for the rapid adaptation of OP-degrading enzymes to changing environmental pressures.

Structural flexibility enhances the reactivity of the bioremediator glycerophosphodiesterase by fine-tuning its mechanism of hydrolysis.

The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) belongs to the family of binuclear metallohydrolases and has attracted recent attention due to its potential in bioremediation. Formation of a catalytically competent binuclear center is promoted by the substrate (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Using the paramagnetic properties of Mn(II), we estimated the K(d) values for the metal ions in the alpha and beta sites to be 29 and 344 microM, respectively, in the absence of a substrate analogue. In its presence, the affinity of the beta site increases substantially (K(d) = 56 microM), while that of the alpha site is not greatly affected (K(d) = 17 microM). Stopped-flow fluorescence measurements identified three distinct phases in the catalytic turnover, associated with the initial binding of substrate to the active site (k(obs1)), the assembly of a catalytically active binuclear center (k(obs2)), and subsequent slower structural rearrangements to optimize catalysis (k(obs3)). These three phases depend on the concentration of substrate ([S]), with k(obs1) and k(obs2) reaching maximum values at high [S] (354 and 38 s(-1), respectively), whereas k(obs3) is reduced as [S] is increased. The k(cat) for the hydrolysis of the substrate bis(para-nitrophenyl) phosphate (approximately 1 s(-1)) gradually increases from the moment of initiating the reaction, reaching a maximum when the structural change associated with k(obs3) is complete. This structural change is mediated via an extensive hydrogen-bond network that connects the coordination sphere with the substrate binding pocket, as demonstrated by mutation of two residues in this network (His81 and His217). The identities of both the substrate and the metal ion also affect interactions within this H-bond network, thus leading to some mechanistic variations. Overall, the mechanism employed by GpdQ is a paradigm of a substrate- and metal-ion-induced fit to optimize catalysis.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase.

BACKGROUND: The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. RESULTS: In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. CONCLUSION: This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents.

Functional characterization by genetic complementation of aroB-encoded dehydroquinate synthase from Mycobacterium tuberculosis H37Rv and its heterologous expression and purification.

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.

Chorismate synthase: an attractive target for drug development against orphan diseases.

The increase in incidence of infectious diseases worldwide, particularly in developing countries, is worrying. Each year, 14 million people are killed by infectious diseases, mainly HIV/AIDS, respiratory infections, malaria and tuberculosis.. Despite the great burden in the poor countries, drug discovery to treat tropical diseases has come to a standstill. There is no interest by the pharmaceutical industry in drug development against the major diseases of the poor countries, since the financial return cannot be guaranteed. This has created an urgent need for new therapeutics to neglected diseases. A possible approach has been the exploitation of the inhibition of unique targets, vital to the pathogen such as the shikimate pathway enzymes, which are present in bacteria, fungi and apicomplexan parasites but are absent in mammals. The chorismate synthase (CS) catalyses the seventh step in this pathway, the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. The strict requirement for a reduced flavin mononucleotide and the anti 1,4 elimination are both unusual aspects which make CS reaction unique among flavin-dependent enzymes, representing an important target for the chemotherapeutic agents development. In this review we present the main biochemical features of CS from bacterial and fungal sources and their difference from the apicomplexan CS. The CS mechanisms proposed are discussed and compared with structural data. The CS structures of some organisms are compared and their distinct features analyzed. Some known CS inhibitors are presented and the main characteristics are discussed. The structural and kinetics data reviewed here can be useful for the design of inhibitors.

Structure of chorismate synthase from Mycobacterium tuberculosis.

In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatics amino acids, are synthesized through seven enzymes from the shikimate pathway, which are absent in mammals. The absence of this pathway in mammals make them potential targets for development of new therapy against infectious diseases, such as tuberculosis, which is the world's second commonest cause of death from infectious disease. The last enzyme of shikimate pathway is the chorismate synthase (CS), which is responsible for conversion of the 5-enolpyruvylshikimate-3-phosphate to chorismate. Here, we report the crystallographic structure of CS from Mycobacterium tuberculosis (MtCS) at 2.65 A resolution. The MtCS structure is similar to other CS structures, presenting beta-alpha-beta sandwich structural topology, in which each monomer of MtCS consists of a central helical core. The MtCS can be described as a tetramer formed by a dimer of dimers. However, analytical ultracentrifugation studies suggest the MtCS is a dimer with a more asymmetric shape than observed on the crystallographic dimer and the existence of a low equilibrium between dimer and tetramer. Our results suggest that the MtCS oligomerization is concentration dependent and some conformational changes must be involved on that event.

DAHP synthase from Mycobacterium tuberculosis H37Rv: cloning, expression, and purification of functional enzyme.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of mortality due to a bacterial pathogen. According to the 2004 Global TB Control Report of the World Health Organization, there are 300,000 new cases per year of multi-drug resistant strains (MDR-TB), defined as resistant to isoniazid and rifampicin, and 79% of MDR-TB cases are now "super strains," resistant to at least three of the four main drugs used to treat TB. Thus there is a need for the development of effective new agents to treat TB. The shikimate pathway is an attractive target for the development of antimycobacterial agents because it has been shown to be essential for the viability of M. tuberculosis, but absent from mammals. The M. tuberculosis aroG-encoded 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (mtDAHPS) catalyzes the first committed step in this pathway. Here we describe the PCR amplification, cloning, and sequencing of aroG structural gene from M. tuberculosis H37Rv. The expression of recombinant mtDAHPS protein in the soluble form was obtained in Escherichia coli Rosetta-gami (DE3) host cells without IPTG induction. An approximately threefold purification protocol yielded homogeneous enzyme with a specific activity value of 0.47U mg(-1) under the experimental conditions used. Gel filtration chromatography results demonstrate that recombinant mtDAHPS is a pentamer in solution. The availability of homogeneous mtDAHPS will allow structural and kinetics studies to be performed aiming at antitubercular agents development.

Crystallization and preliminary X-ray crystallographic analysis of chorismate synthase from Mycobacterium tuberculosis.

The enzymes of the shikimate pathway are potential targets for the development of new therapies because they are essential for bacteria but absent from mammals. The last step in this pathway is performed by chorismate synthase (CS), which catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. Optimization of crystallization trials allowed the crystallization of homogeneous recombinant CS from Mycobacterium tuberculosis (MtCS). The crystals of MtCS belong to space group P6(4)22 (or P6(2)22) and diffract to 2.8 A resolution, with unit-cell parameters a = b = 129.7, c = 156.8 A. There are two molecules in the asymmetric unit. Molecular-replacement trials were not successful. Heavy-atom derivative screening is in progress.