Michael D. Toney

Heavy-enzyme kinetic isotope effects on proton transfer in alanine racemase

The catalytic effects of perdeuterating the pyridoxal phosphate-dependent enzyme alanine racemase from Geobacillus stearothermophilus are reported. The mass of the heavy perdeuterated form is ~5.5% greater than that of the protiated form, causing kinetic isotope effects (KIEs) of ~1.3 on k(cat) and k(cat)/K(M) for both L- and D-alanine. These values increase when Cα-deuterated alanine is used as the substrate. The heavy-enzyme KIEs of ~3 on k(cat)/K(M) with deuterated substrates are greater than the product of the individual heavy-enzyme and primary substrate KIEs. This breakdown of the rule of the geometric mean is likely due to coupled motion between the protein and the proton-transfer reaction coordinate in the rate-limiting step. These data implicate a direct role for protein vibrational motions in barrier crossing for proton-transfer steps in alanine racemase.

Engineering amyloid fibrils from β-solenoid proteins for biomaterials applications.

Nature provides numerous examples of self-assembly that can potentially be implemented for materials applications. Considerable attention has been given to one-dimensional cross-β or amyloid structures that can serve as templates for wire growth or strengthen materials such as glue or cement. Here, we demonstrate controlled amyloid self-assembly based on modifications of β-solenoid proteins. They occur naturally in several contexts (e.g., antifreeze proteins, drug resistance proteins) but do not aggregate in vivo due to capping structures or distortions at their ends. Removal of these capping structures and regularization of the ends of the spruce budworm and rye grass antifreeze proteins yield micron length amyloid fibrils with predictable heights, which can be a platform for biomaterial-based self-assembly. The design process, including all-atom molecular dynamics simulations, purification, and self-assembly procedures are described. Fibril formation with the predicted characteristics is supported by evidence from thioflavin-T fluorescence, circular dichroism, dynamic light scattering, and atomic force microscopy. Additionally, we find evidence for lateral assembly of the modified spruce budworm antifreeze fibrils with sufficient incubation time. The kinetics of polymerization are consistent with those for other amyloid formation reactions and are relatively fast due to the preformed nature of the polymerization nucleus.

1,4,7-Trimethyloxatriquinane: SN2 Reaction at Tertiary Carbon

The synthesis of 1,4,7-trimethyloxatriquinane (1), a 3-fold tertiary alkyl oxonium salt, is described. Compound 1 is inert to solvolysis with alcohols, even at elevated temperatures, but undergoes facile substitution with the strongly nucleophilic azide anion. Since an S(N)1 pathway is excluded, the only reasonable mechanistic interpretation for the reaction between 1 and N(3)(-) is S(N)2, despite the fact that substitution is occurring at a tertiary carbon center. This finding is supported by computational modeling and a study of the reaction kinetics, and is also consistent with observed solvent and salt effects.

Targeting Multiple Chorismate-Utilizing Enzymes with a Single Inhibitor: Validation of a Three-Stage Design

Chorismate-utilizing enzymes are attractive antimicrobial drug targets due to their absence in humans and their central role in bacterial survival and virulence. The structural and mechanistic homology of a group of these inspired the goal of discovering inhibitors that target multiple enzymes. Previously, we discovered seven inhibitors of 4-amino-4-deoxychorismate synthase (ADCS) in an on-bead, fluorescent-based screen of a 2304-member one-bead-one-compound combinatorial library. The inhibitors comprise PAYLOAD and COMBI stages, which interact with active site and surface residues, respectively, and are linked by a SPACER stage. These seven compounds, and six derivatives thereof, also inhibit two other enzymes in this family, isochorismate synthase (IS) and anthranilate synthase (AS). The best binding compound inhibits ADCS, IS, and AS with K(i) values of 720, 56, and 80 microM, respectively. Inhibitors with varying SPACER lengths show the original choice of lysine to be optimal. Lastly, inhibition data confirm the PAYLOAD stage directs the inhibitors to the ADCS active site.

Nucleophile specificity in anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and salicylate synthase.

Anthranilate synthase (AS), aminodeoxychorismate synthase (ADCS), isochorismate synthase (IS), and salicylate synthase (SS) are structurally homologous chorismate-utilizing enzymes that carry out the first committed step in the formation of tryptophan, folate, and the siderophores enterobactin and mycobactin, respectively. Each enzyme catalyzes a nucleophilic substitution reaction, but IS and SS are uniquely able to employ water as a nucleophile. Lys147 has been proposed to be the catalytic base that activates water for nucleophilic attack in IS and SS reactions; in AS and ADCS, glutamine occupies the analogous position. To probe the role of Lys147 as a catalytic base, the K147Q IS, K147Q SS, Q147K AS, and Q147K ADCS mutants were prepared and enzyme reactions were analyzed by high-performance liquid chromatography. Q147K AS employs water as a nucleophile to a small extent, and the cognate activities of K147Q IS and K147Q SS were reduced approximately 25- and approximately 50-fold, respectively. Therefore, Lys147 is not solely responsible for activation of water as a nucleophile. Additional factors that contribute to water activation are proposed. A change in substrate preference for K147Q SS pyruvate lyase activity indicates Lys147 partially controls SS reaction specificity. Finally, we demonstrate that AS, ADCS, IS, and SS do not possess chorismate mutase promiscuous activity, contrary to several previous reports.

Janus: Prediction and Ranking of Mutations Required for Functional Interconversion of Enzymes

Identification of residues responsible for functional specificity in enzymes is a challenging and important problem in protein chemistry. Active-site residues are generally easy to identify, but residues outside the active site are also important to catalysis and their identities and roles are more difficult to determine. We report a method based on analysis of multiple sequence alignments, embodied in our program Janus, for predicting mutations required to interconvert structurally related but functionally distinct enzymes. Conversion of aspartate aminotransferase into tyrosine aminotransferase is demonstrated and compared to previous efforts. Incorporation of 35 predicted mutations resulted in an enzyme with the desired substrate specificity but low catalytic activity. A single round of DNA back-shuffling with wild-type aspartate aminotransferase on this variant generated mutants with tyrosine aminotransferase activities better than those previously realized from rational design or directed evolution. Methods such as this, coupled with computational modeling, may prove invaluable in furthering our understanding of enzyme catalysis and engineering.

Orthogonally protected thiazole and isoxazole diamino acids: an efficient synthetic route

Heterocyclic and heteroaromatic amino acids (HAAs) are central to the motifs of peptide antibiotics, including microcin B17, nostocyclamide, telomestatin, and thiostrepton. aAmino acids undergo cyclization and oxidation to form heteroaromatic rings, notably, thiazoles, oxazoles, indoles, and pyridines, which give rise to well-documented antibiotic activity. Few of these targets have succumbed to total synthesis due, in large part, to the demand for orthogonally protected HAA building blocks. In contrast, commercial orthogonally protected natural amino acids, most commonly lysine and aspartic acid, are routinely used as the branch point in the synthesis of branched or cyclic peptide and oligosaccharide mimetics (Figure 1 a). Similarly, these agents see action in the ligation of imaging agents (Figure 1 b) and in diversity-oriented syntheses (e.g., I!II, Figure 1 c). However, the stringent orthogonal chemistry requirements, especially in solid-phase synthesis, make optimization at this branch-point region challenging. Surprisingly, methods to generate new heterocyclic nonnatural amino acids with an additional orthogonally protected amino group (e.g., diamino acids), are still rare. Nonnatural conformationally restrictive amino acids have potential in the discovery of new peptidomimetics and in efforts to improve the pharmacological and protease resistant properties of bioactive peptides. 9] There is demand for practical HAA syntheses that deliver orthogonally protected diamino acids compatible with the traditional solid and solution phase 9-fluorenylmethoxycarbonyl (Fmoc) protection strategy. Thus our focus herein is on the development of short, high yielding syntheses delivering heteroaromatic monoand diamino acids from readily available starting materials. Herein, we report an efficient synthesis yielding thiazoleand isoxazole-based HAAs from b-amino acids. This strategy allows for orthogonal carbamate protection that permits independent synthetic manipulation (Figure 1). Further, the viability of the synthesized HAAs as branch-point amino acids is demonstrated in the solid-phase synthesis of an inhibitor of two chorismate utilizing enzymes, anthranilate synthase (AS) and isochorismate synthase (IS). This inhibitor shows twoand threefold better activity than its lysine predecessor in the inhibition of AS and IS, respectively. A wide variety of b-amino acids are commercially available and considerable synthetic effort has been focused on producing novel optically active b-amino acids. This availability makes b-amino acids an attractive starting material for this work. As outlined in Figure 2, our synthetic method began by carbamate protection (Teoc, Boc, Cbz, and Alloc) of b-alanine following literature procedures. These protected acids were subjected to coupling conditions to install the Meldrum acid moiety in 94–98 % yield. Intramolecular cyclization of 1 a–d!2 a–d is accomplished quantitatively in EtOAc at reflux via a presumed ketene intermediate. In a modification of Suzuki s general method of cyclocondensa[a] Dr. J. D. Butler, K. C. Coffman, Dr. K. T. Ziebart, Prof. M. D. Toney, Prof. M. J. Kurth Department of Chemistry, University of California, Davis One Shields Avenue, Davis, CA 95616 (USA) Fax: (+1) 530-752-8995 E-mail : mjkurth@ucdavis.edu Homepage: http://chemgroups.ucdavis.edu/~kurth/ Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201001492. Figure 1. Common motifs and methods employing an orthogonally protected diamino acid, which include a) branched peptides, b) ligated imaging agents, and c) diversity-oriented methods.

Ground-state electronic destabilization via hyperconjugation in aspartate aminotransferase.

Binding isotope effects for l-aspartate reacting with the inactive K258A mutant of PLP-dependent aspartate aminotransferase to give a stable external aldimine intermediate are reported. They provide direct evidence for electronic ground-state destabilization via hyperconjugation. The smaller equilibrium isotope effect with deazaPLP-reconstituted K258A indicates that the pyridine nitrogen plays an important role in labilizing the Cα-H bond.

Conversion of Aminodeoxychorismate Synthase into Anthranilate Synthase with Janus Mutations: Mechanism of Pyruvate Elimination Catalyzed by Chorismate Enzymes

The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS.

Mutational analysis of substrate interactions with the active site of dialkylglycine decarboxylase.

Pyridoxal phosphate (PLP)-dependent enzymes catalyze many different types of reactions at the alpha-, beta-, and gamma-carbons of amine and amino acid substrates. Dialkylglycine decarboxylase (DGD) is an unusual PLP-dependent enzyme that catalyzes two reaction types, decarboxylation and transamination, in the same active site. A structurally based, functional model has been proposed for the DGD active site, which maintains that R406 is important in determining substrate specificity through interactions with the substrate carboxylate while W138 provides specificity for short-chain alkyl groups. The mechanistic roles of R406 and W138 were investigated using site-directed mutagenesis, alternate substrates, and analysis of steady-state and half-reaction kinetics. Experiments with the R406M and R406K mutants confirm the importance of R406 in substrate binding. Surprisingly, this work also shows that the positive charge of R406 facilitates catalysis of decarboxylation. The W138F mutant demonstrates that W138 indeed acts to limit the size of the subsite C binding pocket, determining specificity for 2,2-dialkylglycines with small side chains as predicted by the model. Finally, work with the double mutant W138F/M141R shows that these mutations expand substrate specificity to include l-glutamate and lead to an increase in specificity for l-glutamate over 2-aminoisobutyrate of approximately 8 orders of magnitude compared to that of wild-type DGD.