Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis.

The widespread use of scented products causes an increase of allergic contact dermatitis to fragrance compounds in Western countries today. Many fragrance compounds are prone to autoxidation, forming hydroperoxides as their primary oxidation products. Hydroperoxides are known to be strong allergens and to form specific immunogenic complexes. However, the mechanisms for the formation of the immunogenic complexes are largely unknown. We have investigated this mechanism for (5R)-5-isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (Lim-2-OOH) by studying the formation of adducts in the reaction between this hydroperoxide and 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride (Fe(III)TPPCl) in the presence of protected cysteine (NAc-Cys-OMe) or glutathione (GSH). Isolated adducts originate from the addition of the thiol group of NAc-Cys-OMe over the carbon-carbon double bonds of carvone. Furthermore, adducts between NAc-Cys-OMe and carveol as well as between GSH and carvone have been identified. The formation of these adducts most likely proceeds via the radical thiol-ene mechanism. The addition of a terpene moiety to cysteine offers an explanation of the specificity of the immune response to structurally different hydroperoxides. These results also explain the lack of cross-reactivity between carvone and Lim-2-OOH. In conclusion, we propose that immunogenic complexes of olefinic hydroperoxides can be formed via the radical thiol-ene mechanism. These complexes will be specific for the individual olefinic hydroperoxides due to the inclusion of a terpene moiety derived from the hydroperoxide.

No abstract

Weak multivalent interactions are now recognized as key to many biological processes.[1] Since the discovery of carbohydrate-carbohydrate interactions (CCIs), [2] studies of this phenomenon have now linked CCIs (both cis-and transCCIs) to critical biological recognition events, such as cell signaling and adhesion, fertilization, and metastasis.[3] CCIs are intrinsically weak so their study and quantification at the monovalent level is a significant challenge that represents the focus of this Communication.Previous work has relied on macroscopic or multivalent systems including synthetic polymers, [4] micelles and vesicles, [5] glycosylated nanoparticles, [6] and Langmuir-Blodgett monolayers. [7] These studies have generally (though not exclusively) focused on the biologically important Lewis XLewis X (Le X -Le X ) interaction. [4][5][6][7][8] As a result, a number of factors important in CCIs are now apparent, and these include multivalent (Velcro-like) presentation of carbohydrates on a surface; a requirement for polyamphiphilic surfaces associated with the hydraphobic effect; [9] and, in certain cases, roles for both divalent metal cations (e.g. Ca ) and ionic (charge) effects. While multivalency effectively amplifies CCIs, the complexity of such macroscopic systems makes mapping the individual impact of component carbohydrate (CHO) units and their associated molecular features difficult to define.To achieve a more detailed picture of CCIs, while recognizing the inherent challenge of studying this phenomenon in isolation (i.e. outside of a multivalent environment), [10] we have evaluated the ability of a conformationally dynamic system to report on a weak, attractive CCI based on Le X -Le X . Random-coil:a-helix equilibria displayed by alanine-rich peptides in aqueous solution, where helix content is highly sensitive to small changes in the free energy of helix formation, provide an attractive, effective and potentially versatile vehicle for this purpose (Figure 1 a). The requisite peptides are readily accessible, and helix content can be measured accurately by circular dichroism (CD) spectroscopy. We posit that with two CHOs ligated at specified positions (Figure 1 b) on the peptide backbone, perturbation of this highly sensitive equilibrium to a more helical state would indicate the presence of an attractive (i.e. stabilizing) CCI, thereby providing a means of studying this phenomenon in comparative isolation, outside of a multivalent environment.To validate the feasibility of a peptide-based reporter for this purpose, a series of 19-residue host peptides was designed (Figure 1 c). These comprised mainly Ala and Lys residues, incorporating Tyr (as a UV determinant of peptide concen-