Bifunctional catalysis of proton transfer at an antibody active site.
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Antibodies↗
Biomedical subjects
Florian P Seebeck
Publications and source records attributed to Florian P Seebeck.
Stereoselectivity and expanded substrate scope of an engineered PLP-dependent aldolase.
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Aldehyde-Lyases↗
Ribosomal synthesis of dehydroalanine-containing peptides.
Dehydroalanine is a nonproteinogenic amino acid, but it is a component of a wide variety of natural products with therapeutic activities. Indeed, this alpha,beta-unsaturated residue is a highly versatile building block due to its rigidifying effect on peptide backbones and its electrophilicity which allows site-specific thiol ligations of peptides with small molecules or proteins. To harness such versatility in genetically encoded, combinatorial peptide libraries, we report a simple and robust method for the ribosomal synthesis of dehydroalanine-containing peptides. Selenalysine, a selenium-containing lysine analogue, was recruited as a masked dehydroalanine equivalent. This residue is efficiently incorporated by a reconstituted Escherichia coli translation system at high fidelity and efficiency despite the presence of low levels of lysine. Mild oxidative conditions were used to convert selenalysine into dehydroalanine post-translationally. Using this method, we demonstrate the preparation of polyunsaturated and highly decorated peptides. This report is an important step toward the preparation and selection of large libraries of protein-reactive compounds with potential use as novel drugs or as analytical tools.
Alanine↗
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↗
Tunnel plasticity and quaternary structural integrity of a pentameric protein ring.
Cyclic protein oligomers are common in cells. However, the importance of the residues that line the central tunnel of protein rings for overall architectural integrity is not well understood. To investigate the role of tunnel positions in protein assembly and stability, we prepared variants of the homo-pentameric lumazine synthase (LS) from Saccharomyces cerevisiae in which the three residues that line the middle of the tunnel were simultaneously changed. As a consequence of symmetry, these mutations cause a total of 15 changes in the structure of the pentameric complex. Detailed characterization of the variants indicates that they retain quaternary structural integrity, even in cases where the mutations induce considerable secondary structure alterations. The tunnels of symmetric ring-shaped proteins, such as LS, may consequently represent an overlooked site for protein engineering.
Anilino Naphthalenesulfonates↗
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↗
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↗
Conversion of a PLP-dependent racemase into an aldolase by a single active site mutation.
Alanine racemase (Alr) [EC 5.1.1.1] from Geobacillus stearothermophilus is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the first committed step in bacterial cell wall biosynthesis. It is converted to an aldolase upon replacement of Tyr265, which normally serves as a catalytic base in the racemase reaction, with alanine. The Y265A mutation increases catalytic efficiency for cleavage of beta-phenylserine to benzaldehyde and glycine by 2.3 x 105 fold as compared to the wild-type racemase, while racemase activity is greatly decreased. Additional mutagenesis suggests that His166 may act as the base that initiates the retroaldol reaction. The Y265A mutant is highly stereoselective for (2R,3S)-phenylserine, a d-amino acid, and does not process its enantiomer. This preference is consistent with the expected binding mode of substrate in the modified active site and supports the proposal that naturally occurring d-threonine aldolases and alanine racemases derive from a common ancestor.
Alanine Racemase↗