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Biomedical subjects

Donald Hilvert

Publications and source records attributed to Donald Hilvert.

At least 19 recordsLinked to original sources
PubMed2006

A simple tagging system for protein encapsulation.

Molecular containers that encapsulate specific cargo can be useful for many natural and non-natural processes. We report a simple system, based on charge complementarity, for the encapsulation of appropriately tagged proteins within an engineered, proteinaceous capsid. Four negative charges per monomer were added to the lumazine synthase from Aquifex aeolicus (AaLS). The capsids formed by the engineered AaLS associate with green fluorescent protein bearing a positively charged deca-arginine tag upon coproduction in Escherichia coli. Analytical ultracentrifugation and scanning force microscopy studies indicated that the engineered AaLS retains the ability to form capsids, but that their average size was substantially increased. The success of this strategy demonstrates that both the container and guest components of protein-based encapsulation systems can be convergently designed in a straightforward manner, which may help to extend their versatility.

Amino Acid Substitution↗
PubMed2006

Experimental and computational investigation of the uncatalyzed rearrangement and elimination reactions of isochorismate.

The versatile biosynthetic intermediate isochorismate decomposes in aqueous buffer by two competitive pathways, one leading to isoprephenate by a facile Claisen rearrangement and the other to salicylate via elimination of the enolpyruvyl side chain. Computation suggests that both processes are concerted but asynchronous pericyclic reactions, with considerable C-O cleavage in the transition state but relatively little C-C bond formation (rearrangement) or hydrogen atom transfer to the enolpyruvyl side chain (elimination). Kinetic experiments show that rearrangement is roughly 8-times more favorable than elimination. Moreover, transfer of the C2 hydrogen atom to C9 was verified by monitoring the decomposition of [2-(2)H]isochorismate, which was prepared chemoenzymatically from labeled shikimate, by (2)H NMR spectroscopy and observing the appearance of [3-(2)H]pyruvate. Finally, the isotope effects obtained with the C2 deuterated substrate are in good agreement with calculations assuming pericyclic reaction mechanisms. These results provide a benchmark for mechanistic investigations of isochorismate mutase and isochorismate pyruvate lyase, the enzymes that respectively catalyze the rearrangement and elimination reactions in plants and bacteria.

Chorismate Mutase↗
PubMed2005

Isochorismate pyruvate lyase: a pericyclic reaction mechanism?

Isochorismate pyruvate lyase (IPL) catalyzes the cleavage of isochorismate to give salicylate and pyruvate, a key step in bacterial siderophore biosynthesis. We investigated the enzyme from Pseudomonas aeruginosa using isochorismate selectively deuterated at C2 as a substrate. Monitoring the reaction by 2H NMR spectroscopy revealed that the label is quantitatively transferred from C2 to C9, producing stoichiometric amounts of [3-2H]pyruvate as product. Moreover, the deuterium kinetic isotope effect of 2.34 +/- 0.08 on kcat indicates that C-H cleavage is significantly rate limiting. Consistent with these data, hybrid density functional theory (HDFT) calculations at the Becke3LYP/DZ+(2d,p) level of theory predict a concerted but highly asynchronous pericyclic transition structure, in which carbon-oxygen bond cleavage is more advanced than hydrogen atom transfer from C2 to C9; the calculated 2H isotope effect of 2.22 at C2 is in excellent accord with the experimental value. Together, these findings indicate that IPL should be added to the small set of proteins that are known to catalyze pericyclic reactions. They also raise the possibility that enzymes, such as chorismate pyruvate lyase, salicylate synthase, 4-amino-4-deoxychorismate lyase, and anthranilate synthase, which accelerate formally similar reaction steps, may also exploit pericyclic mechanisms.

Binding Sites↗
PubMed2005

Isotope effects on the enzymatic and nonenzymatic reactions of chorismate.

The important biosynthetic intermediate chorismate reacts thermally by two competitive pathways, one leading to 4-hydroxybenzoate via elimination of the enolpyruvyl side chain, and the other to prephenate by a facile Claisen rearrangement. Measurements with isotopically labeled chorismate derivatives indicate that both are concerted sigmatropic processes, controlled by the orientation of the enolpyruvyl group. In the elimination reaction of [4-2H]chorismate, roughly 60% of the label was found in pyruvate after 3 h at 60 degrees C. Moreover, a 1.846 +/- 0.057 2H isotope effect for the transferred hydrogen atom and a 1.0374 +/- 0.0005 18O isotope effect for the ether oxygen show that the transition state for this process is highly asymmetric, with hydrogen atom transfer from C4 to C9 significantly less advanced than C-O bond cleavage. In the competing Claisen rearrangement, a very large 18O isotope effect at the bond-breaking position (1.0482 +/- 0.0005) and a smaller 13C isotope effect at the bond-making position (1.0118 +/- 0.0004) were determined. Isotope effects of similar magnitude characterized the transformations catalyzed by evolutionarily unrelated chorismate mutases from Escherichia coli and Bacillus subtilis. The enzymatic reactions, like their solution counterpart, are thus concerted [3,3]-sigmatropic processes in which C-C bond formation lags behind C-O bond cleavage. However, as substantially larger 18O and smaller 13C isotope effects were observed for a mutant enzyme in which chemistry is fully rate determining, the ionic active site may favor a somewhat more polarized transition state than that seen in solution.

Chorismic Acid↗
PubMed2005

An active enzyme constructed from a 9-amino acid alphabet.

Nature employs a set of 20 amino acids to produce a repertoire of protein structures endowed with sophisticated functions. Here, we combined design and selection to create an enzyme composed entirely from a set of only 9 amino acids that can rescue auxotrophic cells lacking chorismate mutase. The simplified protein captures key structural features of its natural counterpart but appears to be somewhat less stable and more flexible. The potential of a dramatically reduced amino acid alphabet to produce an active catalyst supports the notion that primordial enzymes may have possessed low amino acid diversity and suggests that combinatorial engineering strategies, such as the one used here, may be generally applied to create enzymes with novel structures and functions.

Amino Acid Sequence↗
PubMed2005

Mechanistic insights into the isochorismate pyruvate lyase activity of the catalytically promiscuous PchB from combinatorial mutagenesis and selection.

PchB from Pseudomonas aeruginosa possesses isochorismate pyruvate lyase (IPL) and weak chorismate mutase (CM) activity. Homology modeling based on a structurally characterized CM, coupled with randomization of presumed key active site residues (Arg54, Glu90, Gln91) and in vivo selection for CM activity, was used to derive mechanistic insights into the IPL activity of PchB. Mutation of Arg54 was incompatible with viability, and the CM and IPL activities of an engineered R54K variant were reduced 1,000-fold each. The observation that position 90 was tolerant to substitution but position 91 was essentially confined to Gln or Glu in functional variants rules out involvement of Glu90 in general base catalysis. Counter to the generally accepted mechanistic hypothesis for pyruvate lyases, we propose for PchB a rare [1,5]-sigmatropic reaction mechanism that invokes electrostatic catalysis in analogy to the [3,3]-pericyclic rearrangement of chorismate in CMs. A common catalytic principle for both PchB functions is also supported by the covariance of the catalytic parameters for the CM and IPL activities and the shared functional requirement for a protonated Glu91 in Q91E variants. The experiments demonstrate that focusing directed evolution strategies on the readily accessible surrogate activity of an enzyme can provide valuable insights into the mechanism of the primary reaction.

Amino Acid Sequence↗
PubMed2005

Simultaneous optimization of enzyme activity and quaternary structure by directed evolution.

Natural evolution has produced efficient enzymes of enormous structural diversity. We imitated this natural process in the laboratory to augment the efficiency of an engineered chorismate mutase with low activity and an unusual hexameric topology. By applying two rounds of DNA shuffling and genetic selection, we obtained a 400-fold more efficient enzyme, containing three non-active-site mutations. Detailed biophysical characterization of the evolved variant suggests that it exists predominantly as a trimer in solution, but is otherwise similarly stable as the parent hexamer. The dramatic structural and functional effects achieved by a small number of seemingly innocuous substitutions highlights the utility of directed evolution for modifying protein-protein interactions to produce novel quaternary states with optimized activities.

Chorismate Mutase↗
PubMed2005

Investigation of ligand binding and protein dynamics in Bacillus subtilis chorismate mutase by transverse relaxation optimized spectroscopy-nuclear magnetic resonance.

The structural and dynamical consequences of ligand binding to a monofunctional chorismate mutase from Bacillus subtilis have been investigated by solution NMR spectroscopy. TROSY methods were employed to assign 98% of the backbone (1)H(N), (1)H(alpha), (15)N, (13)C', and (13)C(alpha) resonances as well as 86% of the side chain (13)C resonances of the 44 kDa trimeric enzyme at 20 degrees C. This information was used to map chemical shift perturbations and changes in intramolecular mobility caused by binding of prephenate or a transition state analogue to the X-ray structure. Model-free interpretation of backbone dynamics for the free enzyme and its complexes based on (15)N relaxation data measured at 600 and 900 MHz showed significant structural consolidation of the protein in the presence of a bound ligand. In agreement with earlier structural and biochemical studies, substantial ordering of 10 otherwise highly flexible residues at the C-terminus is particularly notable. The observed changes suggest direct contact between this protein segment and the bound ligand, providing support for the proposal that the C-terminus can serve as a lid for the active site, limiting diffusion into and out of the pocket and possibly imposing conformational control over substrate once bound. Other regions of the protein that experience substantial ligand-induced changes also border the active site or lie along the subunit interfaces, indicating that the enzyme adapts dynamically to ligands by a sort of induced fit mechanism. It is believed that the mutase-catalyzed chorismate-to-prephenate rearrangement is partially encounter controlled, and backbone motions on the millisecond time scale, as seen here, may contribute to the reaction barrier.

Bacillus subtilis↗
PubMed2005

Use of molecular dynamics in the design and structure determination of a photoinducible beta-hairpin.

The study presented here consists of three parts. In the first, the ability of a set of differently substituted diazobenzene-based linkers to act as photoswitchable beta-turn building blocks was assessed. A 12-residue peptide known to form beta-hairpins was taken as the basis for the modeling process. The central (beta-turn) residue pair was successively replaced by six symmetrically ((o,o), (m,m), or (p,p)) substituted (aminomethyl/carboxymethyl or aminoethyl/carboxyethyl) diazobenzene derivatives leading to a set of peptides with a photoswitchable backbone conformation. The folding behavior of each peptide was then investigated by performing molecular dynamics simulations in water (4 ns) and in methanol (10 ns) at room temperature. The simulations suggest that (o,o)- and (m,m)-substituted linkers with a single methylene spacer are significantly better suited to act as photoswitchable beta-turn building blocks than the other linkers examined in this study. The peptide containing the (m,m)-substituted linker was synthesized and characterized by NMR in its cis configuration. In the second part of this study, the structure of this peptide was refined using explicit-solvent simulations and NOE distance restraints, employing a variety of refinement protocols (instantaneous and time-averaged restraining as well as unrestrained simulations). We show that for this type of systems, even short simulations provide a significant improvement in our understanding of their structure if physically meaningful force fields are employed. In the third part, unrestrained explicit-solvent simulations starting from either the NMR model structure (75 ns) or a fully extended structure (25 ns) are shown to converge to a stable beta-hairpin. The resulting ensemble is in good agreement with experimental data, indicating successful structure prediction of the investigated hairpin by classical explicit-solvent molecular dynamics simulations.

Journal Article↗
PubMed2005

Structural origins of efficient proton abstraction from carbon by a catalytic antibody.

Antibody 34E4 catalyzes the conversion of benzisoxazoles to salicylonitriles with high rates and multiple turnovers. The crystal structure of its complex with the benzimidazolium hapten at 2.5-angstroms resolution shows that a combination of hydrogen bonding, pi stacking, and van der Waals interactions is exploited to position both the base, Glu(H50), and the substrate for efficient proton transfer. Suboptimal placement of the catalytic carboxylate, as observed in the 2.8-angstroms structure of the Glu(H50)Asp variant, results in substantially reduced catalytic efficiency. In addition to imposing high positional order on the transition state, the antibody pocket provides a highly structured microenvironment for the reaction in which the carboxylate base is activated through partial desolvation, and the highly polarizable transition state is stabilized by dispersion interactions with the aromatic residue Trp(L91) and solvation of the leaving group oxygen by external water. The enzyme-like efficiency of general base catalysis in this system directly reflects the original hapten design, in which a charged guanidinium moiety was strategically used to elicit an accurately positioned functional group in an appropriate reaction environment and suggests that even larger catalytic effects may be achievable by extending this approach to the induction of acid-base pairs capable of bifunctional catalysis.

Amino Acid Substitution↗
PubMed2005

A photoinducible beta-hairpin.

A photochromic azobenzene linker was incorporated as a turn element into an amino acid sequence known to fold into a beta-hairpin structure in aqueous solution. Oligomer formation when the linker was in its thermodynamically favored trans form prohibited structure determination. Light-induced conformational change of the linker to the cis form led to the formation of monomers which exhibited a well-defined beta-hairpin structure as determined by (1)H NMR. The rate of the light-induced cis-to-trans isomerization of the azobenzene-containing peptide was 30% slower compared to the unsubstituted chromophore. These results suggest that suitably substituted azobenzenes can be used as photoinducible turn elements to investigate and control the folding and stability of beta-sheets.

Azo Compounds↗
PubMed2005

Positional ordering of reacting groups contributes significantly to the efficiency of proton transfer at an antibody active site.

Catalytic antibody 34E4 accelerates the conversion of benzisoxazoles to salicylonitriles with surprising efficiency, exploiting a carboxylate base with an elevated pKa for proton abstraction. Mutagenesis of this antibody, produced as a chimeric Fab, confirms the prediction of a homology model that GluH50 is the essential catalytic residue. Replacement of this residue by glutamine, alanine, or glycine reduces catalytic activity by more than 2.6 x 10(4)-fold. By comparing the chemical proficiencies of the parent antibody with the chemical proficiencies of acetate and the mutants, the effective concentration of the catalytic side chain was estimated to be >51 000 M. The 2.1 kcal/mol destabilization of the transition state observed when GluH50 is replaced by aspartate suggests that positional ordering imposed by the antibody active site contributes significantly to the efficiency of proton transfer. The observation that the GluH50Ala and GluH50Gly variants could not be chemically rescued by exogenous addition of high concentrations of formate or acetate further underscores the advantage the antibody derives from covalently fixing its base at the active site. Although medium effects also play an important role in 34E4, for example in enhancing the reactivity of the carboxylate side chain through desolvation, comparison of 34E4 with less proficient antibodies shows that positioning a carboxylate in a hydrophobic binding pocket alone is insufficient for efficient general base catalysis. Our results demonstrate that structural complementarity between the antibody and its substrate in the transition state is an important and necessary component of 34E4's high activity. By harnessing an additional catalytic group that could serve as a general acid to stabilize developing negative charge in the leaving group, overall efficiencies rivaling those of highly evolved enzymes should be accessible.

Animals↗
PubMed2005

Characterization of the secreted chorismate mutase from the pathogen Mycobacterium tuberculosis.

The gene encompassing ORF Rv1885c with weak sequence similarity to AroQ chorismate mutases (CMs) was cloned from the genome of Mycobacterium tuberculosis and expressed in Escherichia coli. The gene product (*MtCM) complements a CM-deficient E. coli strain, but only if produced without the predicted N-terminal signal sequence typical of M. tuberculosis. The mature *MtCM, which was purified by exploiting its resistance to irreversible thermal denaturation, possesses high CM activity in vitro. The enzyme follows simple Michaelis-Menten kinetics, having a k(cat) of 50 s(-1) and a K(m) of 180 microM (at 30 degrees C and pH 7.5). *MtCM was shown to be a dimer by analytical ultracentrifugation and size-exclusion chromatography. Secondary-structure prediction and CD spectroscopy confirmed that *MtCM is a member of the all-alpha-helical AroQ class of CMs, but it seems to have a topologically rearranged AroQ fold. Because CMs are normally intracellular metabolic enzymes required for the biosynthesis of phenylalanine and tyrosine, the existence of an exported CM in Gram-positive M. tuberculosis is puzzling. The observation that homologs of *MtCM with a predicted export sequence are generally only present in parasitic or pathogenic organisms suggests that secreted CMs may have evolved to participate in some aspect of parasitism or pathogenesis yet to be unraveled.

Amino Acid Sequence↗
PubMed2004

An enzymatic molten globule: efficient coupling of folding and catalysis.

A highly active, monomeric chorismate mutase, obtained by topological redesign of a dimeric helical bundle enzyme from Methanococcus jannaschii, was investigated by NMR and various other biochemical techniques, including H/D exchange. Although structural disorder is generally considered to be incompatible with efficient catalysis, the monomer, unlike its natural counterpart, unexpectedly possesses all of the characteristics of a molten globule. Global conformational ordering, observed upon binding of a transition state analog, indicates that folding can be coupled to catalysis with minimal energetic penalty. These results support the suggestion that many modern enzymes might have evolved from molten globule precursors. Insofar as their structural plasticity confers relaxed substrate specificity and/or catalytic promiscuity, molten globules may also be attractive starting points for the evolution of new catalysts in the laboratory.

Chorismate Mutase↗
PubMed2004

Nonspecific medium effects versus specific group positioning in the antibody and albumin catalysis of the base-promoted ring-opening reactions of benzisoxazoles.

The mechanisms by which solvents, antibodies, and albumins influence the rates of base-catalyzed reactions of benzisoxazoles have been explored theoretically. New experimental data on substituent effects and rates of reactions in several solvents, in an antibody, and in an albumin are reported. Quantum mechanical calculations were carried out for the reactions in water and acetonitrile, and docking of the transition state into a homology model of antibody 34E4 and an X-ray structure of human serum albumin was accomplished. A microenvironment made up of catalytic polar groups (glutamate in antibody 34E4 and lysine in human serum albumin) surrounded by relatively nonpolar groups is present in both catalytic proteins.

Antibodies, Catalytic↗
PubMed2004

Immunological optimization of a generic hydrophobic pocket for high affinity hapten binding and Diels-Alder activity.

Antibody 1E9, which binds a tetrachloronorbornene derivative with subnanomolar affinity and catalyzes the Diels-Alder reaction between tetrachlorothiophene dioxide and N-ethylmaleimide with high efficiency, arose from a family of highly restricted germ-line immunoglobulins that bind diverse hydrophobic ligands. Two somatic mutations, one at position L89 in the light chain (SerL89Phe) and another at position H47 in the heavy chain (TrpH47Leu), have been postulated to be responsible for the unusually high degree of shape and chemical complementarity observed in the crystal structure of 1E9 complexed with its hapten. To test this hypothesis, the germ-line sequence at these two positions was restored by site-directed mutagenesis. The ensuing 160 to 3900-fold decrease in hapten affinity and the complete loss of catalytic activity support the hypothesis that these somatic mutations substantially remodel the antibody binding pocket. Mutation of the highly conserved hydrogen-bond donor AsnH35, which sits at the bottom of the active site and is a hallmark of this family of antibodies, is also catastrophic with respect to hapten binding and catalysis. In contrast, residues in the CDR H3 loop, which contributes a significant fraction of the hapten-contacting protein surface, have a more subtle influence on the properties of 1E9. Interestingly, while most changes in this loop have neutral or modestly deleterious effects, replacement of MetH100b at the floor of the pocket with phenylalanine leads to a significant sevenfold increase in catalytic activity. The latter result is surprising given the unusually close fit of the parent antibody to the transition-state analogue. Further fine-tuning of the interactions between 1E9 and its ligands by introducing mutations outside the active site could conceivably yield substantially more active catalysts.

Amino Acid Substitution↗
PubMed2004

Deciphering enzymes. Genetic selection as a probe of structure and mechanism.

The efficient engineering of enzymes with novel activities remains an ongoing challenge. Towards this end, genetic selection techniques provide a method for finding rare solutions to catalytic problems that requires only a limited foreknowledge of structure-function relationships. We have used genetic selections to extensively probe the structure and mechanism of chorismate mutases. The insights gained from these investigations will aid future enzyme design efforts.

Binding Sites↗