Panoramic view of a superfamily of phosphatases through substrate profiling.

Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.

Chemo- and Regioselective Organo-Photoredox Catalyzed Hydroformylation of Styrenes via a Radical Pathway.

An unprecedented, chemo- and regioselective, organo-photoredox catalyzed hydroformylation reaction of aryl olefins with diethoxyacetic acid as the formylation reagent is described. In contrast to traditional transition metal promoted ionic hydroformylation reactions, the new process follows a unique photoredox promoted, free radical pathway. In this process, a formyl radical equivalent, produced from diethoxacetic acid through a dye (4CzIPN) photocatalyzed, sequential oxidation-decarboxylation route, regio- and chemoselectively adds to a styrene substrate. Importantly, under the optimized reaction conditions the benzylic radical formed in this manner is reduced by SET from the anion radical of 4CzIPN to generate a benzylic anion. Finally, protonation produces the hydroformylation product. By using the new protocol, aldehydes can be generated regioselectively in up to 90% yield. A broad array of functional groups is tolerated in the process, which takes place under mild, metal-free conditions.

Catalytic Mechanism of the Hotdog-Fold Thioesterase PA1618 Revealed by X-ray Structure Determination of a Substrate-Bound Oxygen Ester Analogue Complex.

Thioesterase activity accounts for the majority of the activities in the hotdog-fold superfamily. The structures and mechanisms of catalysis for many hotdog enzymes have been elucidated by X-ray crystallography and kinetics to probe the specific substrate usage and cellular functions. However, structures of hotdog thioesterases in complexes with substrate analogues reported to date utilize ligands that either represent truncations of the substrate or include additional atoms to prevent hydrolysis. Here we present the synthesis of an isosteric and isoelectronic substrate analogue-benzoyl-OdCoA-and the X-ray crystal structure of a complex of the analogue with Pseudomonas aeruginosa hotdog thioesterase PA1618 (at 1.72 Šresolution). The complex is compared with that of the "imperfect" substrate analogue phenacyl-CoA, refined to a resolution of 1.62 Å. Kinetic and structural results are consistent with Glu64 as the catalytic residue and with the involvement of Gln49 in stabilization of the transition state. Structural comparison of the two ligand-bound structures revealed a crucial ordered water molecule coordinated in the active site of the benzoyl-OdCoA structure but not present in the phenacyl-CoA-bound structure. This suggests a general base mechanism of catalysis in which Glu64 activates the coordinated water nucleophile. Together, our findings reveal the importance of a closely similar substrate analogue to determine the true substrate binding and catalytic mechanism.

Visible-Light-Promoted Nickel- and Organic-Dye-Cocatalyzed Formylation Reaction of Aryl Halides and Triflates and Vinyl Bromides with Diethoxyacetic Acid as a Formyl Equivalent.

A simple formylation reaction of aryl halides, aryl triflates, and vinyl bromides under synergistic nickel- and organic-dye-mediated photoredox catalysis is reported. Distinct from widely used palladium-catalyzed formylation processes, this reaction proceeds by a two-step mechanistic sequence involving initial in situ generation of the diethoxymethyl radical from diethoxyacetic acid by a 4CzIPN-mediated photoredox reaction. The formyl-radical equivalent then undergoes nickel-catalyzed substitution reactions with aryl halides and triflates and vinyl bromides to form the corresponding aldehyde products. Significantly, besides aryl bromides, less reactive aryl chlorides and triflates and vinyl halides serve as effective substrates for this process. Since the mild conditions involved in this reaction tolerate a plethora of functional groups, the process can be applied to the efficient preparation of diverse aromatic aldehydes.

Synthesis of Aldehydes by Organocatalytic Formylation Reactions of Boronic Acids with Glyoxylic Acid.

Reported herein is a conceptually novel organocatalytic strategy for the formylation of boronic acids. New reactivity is engineered into the α-amino-acid-forming Petasis reaction occurring between aryl boronic acids, amines, and glyoxylic acids to prepare aldehydes. The operational simplicity of the process and its ability to generate structurally diverse and valued aryl, heteroaryl, and α,ß-unsaturated aldehydes containing a wide array of functional groups, demonstrates the practical utility of the new synthetic strategy.

Single Electron Transfer-Promoted Photochemical Reactions of Secondary N-Trimethylsilylmethyl-N-benzylamines Leading to Aminomethylation of Fullerene C60.

Photoreactions between C60 and secondary N-trimethylsilylmethyl-N-benzylamines were explored to evaluate the feasibility of a new method for secondary aminomethylation of electron acceptors. The results show that photoreactions of C60 with these secondary amines in 10% EtOH-toluene occur to form aminomethyl-1,2-dihydrofullerenes predominantly through a pathway involving single electron transfer (SET)-promoted formation of secondary aminium radicals followed by preferential loss of the α-trimethylsilyl group. The aminomethyl radicals formed in this manner then couple with C60 or C60(•-) to form radical or anion precursors of the aminomethyl-1,2-dihydrofullerenes. In contrast to thermal and photochemical strategies developed previously, the new SET photochemical approach using α-trimethylsilyl-substituted secondary amines is both mild and efficient, and as a result, it should be useful in broadening the library of substituted fullerenes. Moreover, the results should have an impact on the design of SET-promoted C-C bond forming reactions. Specifically, introduction of an α-trimethylsilyl group leads to a change in the chemoselectivity of SET-promoted reactions of secondary amines with acceptors that typically favor aminium radical N-H deprotonation, leading to N-C bond formation. Finally, symmetric and unsymmetric fulleropyrrolidines are also generated in yields that are highly dependent on the electronic properties of arene ring substituents in amines, irradiation time, and solvent.

Single electron transfer promoted photoaddition reactions of α-trimethylsilyl substituted secondary N-alkylamines with fullerene C60.

Single electron transfer (SET) promoted photoaddition reactions of secondary N-α-trimethylsilyl-N-alkylamines to C60 were explored to gain a deeper understanding of the mechanistic pathways followed and to expand the library of novel types of organofullerenes that can be generated using this approach. The results show that photoreactions of 10% EtOH-toluene solutions containing C60 and N-α-trimethylsilyl-N-alkylamines produce either aminomethyl-1,2-dihydrofullerenes or symmetric fulleropyrrolidines as major products depending on the nature of alkyl substituents. In contrast, photoreactions of 10% EtOH-ODCB solutions of these amines with C60 mainly lead to the formation of symmetric fulleropyrrolidines. Based on the analysis of product distributions and the results of earlier studies, two feasible mechanistic pathways are proposed for these processes. One route is initiated by SET from the amine substrates to the triplet-excited state of C60 to form the corresponding aminium radicals and C60 anion radicals. EtOH-promoted desilylation of the aminium radicals then takes place to produce aminomethyl radicals which can either add to C60 or couple with the C60 radical anions to form respective radicals or anion precursors of aminomethyl-1,2-dihydrofullerene products. The competing pathway leading to the generation of symmetric fulleropyrrolidines also involves the formation of aminomethyl radicals by using the sequential SET-desilylation process. In this route, the aminomethyl radicals are oxidized by SET to C60 to form iminium ions, which are then transformed to azomethine ylides by a pathway involving a second molecule of the secondary amine. Dipolar cycloaddition of the azomethine ylides to C60 forms the symmetric fulleropyrrolidine cycloadducts. Importantly, the observation that symmetric fulleropyrrolidines are the sole products formed in photoreactions between N-α-trimethylsilyl-N-alkylamines and C60 in 10% EtOH-ODCB has synthetic significance.

Method for the synthesis of amine-functionalized fullerenes involving SET-promoted photoaddition reactions of α-silylamines.

A novel method for the preparation of structurally diverse fullerene derivatives, which relies on the use of single electron transfer (SET)-promoted photochemical reactions between fullerene C60 and α-trimethylsilylamines, has been developed. Photoirradiation of 10% EtOH-toluene solutions containing C60 and α-silylamines leads to high-yielding, regioselective formation of 1,2-adducts that arise through a pathway in which sequential SET-desilylation occurs to generate α-amino and C60 anion radical pair intermediates, which undergo C-C bond formation. Protonation of generated α-aminofullerene anions gives rise to formation of monoaddition products that possess functionalized α-aminomethyl-substituted 1,2-dihydrofullerene structures. Observations made in this effort show that the use of EtOH in the solvent mixture is critical for efficient photoproduct formation. In contrast to typical thermal and photochemical strategies devised previously for the preparation of fullerene derivatives, the new photochemical approach takes place under mild conditions and does not require the use of excess amounts of substrates. Thus, the method developed in this study could broaden the scope of fullerene chemistry by providing a simple photochemical strategy for large-scale preparation of highly substituted fullerene derivatives. Finally, the α-aminomethyl-substituted 1,2-dihydrofullerene photoadducts are observed to undergo photoinduced fragmentation reactions to produce C60 and the corresponding N-methylamines.

Direct and indirect single electron transfer (SET)-photochemical approaches for the preparation of novel phthalimide and naphthalimide-based lariat-type crown ethers.

In this review, we describe direct and indirect photochemical approaches that have been developed for the preparation of phthalimide- and naphthalimide-based, lariat-type crown ethers. The direct route utilizes a strategy in which nitrogen-linked side chains containing polyethoxy-tethered phthalimides and naphthalimides, possessing terminal α-trialkylsilyl groups, are synthesized utilizing concise routes and UV-irradiation to form macrocyclic ring systems. In contrast, the indirect route developed for the synthesis of lariat-type crown ethers employs sequences in which SET-promoted macrocyclization reactions of α-trialkylsilyl-terminated, polyethoxy-tethered phthalimides and naphthalimides are followed by a side chain introduction through substitution reactions at the amidol centers in the macrocyclic ethers. The combined observations made in these investigations demonstrate the unique features of SET-promoted photocyclization reactions that make them well-suited for the use in the synthesis of functionalized crown ethers. In addition, while some limitations exist for the general use of SET-photochemical reactions in large-scale organic synthesis, important characteristics of the photoinduced macrocyclization reactions make them applicable to unique situations in which high temporal and spatial control is required.

Design, synthesis, and evaluation of inhibitors of pyruvate phosphate dikinase.

Pyruvate phosphate dikinase (PPDK) catalyzes the phosphorylation reaction of pyruvate that forms phosphoenolpyruvate (PEP) via two partial reactions: PPDK + ATP + P(i) → PPDK-P + AMP + PP(i) and PPDK-P + pyruvate → PEP + PPDK. Based on its role in the metabolism of microbial human pathogens, PPDK is a potential drug target. A screen of substances that bind to the PPDK ATP-grasp domain active site revealed that flavone analogues are potent inhibitors of the Clostridium symbiosum PPDK. In silico modeling studies suggested that placement of a 3­6 carbon-tethered ammonium substituent at the 3'- or 4'-positions of 5,7-dihydroxyflavones would result in favorable electrostatic interactions with the PPDK Mg-ATP binding site. As a result, polymethylene-tethered amine derivatives of 5,7-dihydroxyflavones were prepared. Steady-state kinetic analysis of these substances demonstrates that the 4'-aminohexyl-5,7-dyhydroxyflavone 10 is a potent competitive PPDK inhibitor (K(i) = 1.6 ± 0.1 µM). Single turnover experiments were conducted using 4'-aminopropyl-5,7-dihydroxyflavone 7 to show that this flavone specifically targets the ATP binding site and inhibits catalysis of only the PPDK + ATP + P(i) → PPDK-P + AMP PP(i) partial reaction. Finally, the 4'-aminopbutyl-5,7-dihydroxyflavone 8 displays selectivity for inhibition of PPDK versus other enzymes that utilize ATP and NAD.

Effects of alkoxy groups on arene rings of lignin ß-O-4 model compounds on the efficiencies of single electron transfer-promoted photochemical and enzymatic C-C Bond Cleavage Reactions.

To gain information about how alkoxy substitution in arene rings of ß-O-4 structural units within lignin governs the efficiencies/rates of radical cation C1-C2 bond cleavage reactions, single electron transfer (SET) photochemical and lignin peroxidase-catalyzed oxidation reactions of dimeric/tetrameric model compounds have been explored. The results show that the radical cations derived from less alkoxy-substituted dimeric ß-O-4 models undergo more rapid C1-C2 bond cleavage than those of more alkoxy-substituted analogues. These findings gained support from the results of DFT calculations, which demonstrate that C1-C2 bond dissociation energies of ß-O-4 radical cations decrease as the degree of alkoxy substitution decreases. In SET reactions of tetrameric compounds consisting of two ß-O-4 units, containing different degrees of alkoxy substitution, regioselective radical cation C-C bond cleavage was observed to occur in one case at the C1-C2 bond in the less alkoxy-substituted ß-O-4 moiety. However, regioselective C1-C2 cleavage in the more alkoxy-substituted ß-O-4 moiety was observed in another case, suggesting that other factors might participate in controlling this process. These observations show that lignins containing greater proportions of less rather than more alkoxylated rings as part of ß-O-4 units would be more efficiently cleaved by SET mechanisms.

Studies leading to the development of a single-electron transfer (SET) photochemical strategy for syntheses of macrocyclic polyethers, polythioethers, and polyamides.

Organic photochemists began to recognize in the 1970s that a new mechanistic pathway involving excited-state single-electron transfer (SET) could be used to drive unique photochemical reactions. Arnold's seminal studies demonstrated that SET photochemical reactions proceed by way of ion radical intermediates, the properties of which govern the nature of the ensuing reaction pathways. Thus, in contrast to classical photochemical reactions, SET-promoted excited-state processes are controlled by the nature and rates of secondary reactions of intermediate ion radicals. In this Account, we discuss our work in harnessing SET pathways for photochemical synthesis, focusing on the successful production of macrocyclic polyethers, polythioethers, and polyamides. One major thrust of our studies in SET photochemistry has been to develop new, efficient reactions that can be used for the preparation of important natural and non-natural substances. Our efforts with α-silyl donor-tethered phthalimides and naphthalimides have led to the discovery of efficient photochemical processes in which excited-state SET is followed by regioselective formation of carbon-centered radicals. The radical formation takes place through nucleophile-assisted desilylation of intermediate α-silyl-substituted ether-, thioether-, amine-, and amide-centered cation radicals. Early laser flash photolysis studies demonstrated that the rates of methanol- and water-promoted bimolecular desilylations of cation radicals (derived from α-silyl electron donors) exceeded the rates of other cation radical α-fragmentation processes, such as α-deprotonation. In addition, mechanistic analyses of a variety of SET-promoted photocyclization reactions of α-silyl polydonor-linked phthalimides and naphthalimides showed that the chemical and quantum efficiencies of the processes are highly dependent on the lengths and types of the chains connecting the imide acceptor and α-silyl electron donor centers. We also observed that reaction efficiencies are controlled by the rates of desilylation at the α-silyl donor cation radical moieties in intermediate zwitterionic biradicals that are formed by either direct excited-state intramolecular SET or by SET between the donor sites in the intervening chains. It is important to note that knowledge about how these factors govern product yields, regiochemical selectivities, and quantum efficiencies was crucial for the design of synthetically useful photochemical reactions of linked polydonor-acceptor substrates. The fruits of these insights are exemplified by synthetic applications in the concise preparation of cyclic peptide mimics, crown ethers and their lariat- and bis-analogs, and substances that serve as fluorescence sensors for important heavy metal cations.

Photoaddition reactions of acetylpyridines with silyl ketene acetals: SET vs [2 + 2]-cycloaddition pathways.

Photoaddition reactions of silyl ketene acetals with 2-, 3- and 4-acetylpyridine have been explored. The results show that the acetylpyridines react with an electron rich, dimethyl-substituted silyl ketene acetal via a pathway in which excited state single electron transfer (SET) takes place to produce ß-hydroxyesters in high yields. In contrast, photochemical reactions of the acetylpyridines with an electron deficient, nonmethyl-substituted silyl ketene acetal generate oxetanes as major products, which arise via a route involving excited state [2 + 2]-cycloaddition. In addition, an increase in solvent polarity significantly enhances the relative efficiencies of the SET processes versus [2 + 2]-cycloaddition reactions. Importantly, the carbonyl groups rather than the pyridine moieties in the acetylpyridine substrates participate in both types of addition reactions. Finally, the results demonstrate that photoinduced electron transfer (PET)-promoted chemical reactions between acetylpyridines and electron rich silyl ketene acetals in polar solvent serve as useful methods to promote ß-hydroxyester forming, Claisen or Mukaiyama condensation reactions under mild conditions.

Regioselectivity of enzymatic and photochemical single electron transfer promoted carbon-carbon bond fragmentation reactions of tetrameric lignin model compounds.

New types of tetrameric lignin model compounds, which contain the common ß-O-4 and ß-1 structural subunits found in natural lignins, have been prepared and carbon-carbon bond fragmentation reactions of their cation radicals, formed by photochemical (9,10-dicyanoanthracene) and enzymatic (lignin peroxidase) SET-promoted methods, have been explored. The results show that cation radical intermediates generated from the tetrameric model compounds undergo highly regioselective C-C bond cleavage in their ß-1 subunits. The outcomes of these processes suggest that, independent of positive charge and odd-electron distributions, cation radicals of lignins formed by SET to excited states of sensitizers or heme-iron centers in enzymes degrade selectively through bond cleavage reactions in ß-1 vs ß-O-4 moieties. In addition, the findings made in the enzymatic studies demonstrate that the sterically large tetrameric lignin model compounds undergo lignin peroxidase-catalyzed cleavage via a mechanism involving preliminary formation of an enzyme-substrate complex.

Exploration of photochemical reactions of N-trimethylsilylmethyl-substituted uracil, pyridone, and pyrrolidone derivatives.

Photochemical reactions of N-trimethylsilylmethyl-substituted uracil, pyridone and pyrrolidone derivatives were carried out to determine if silicone containing substituents have an impact on excited state reaction profiles. The results show that ultraviolet irradiation of N-trimethylsilylmethyl substituted uracils in the presence of substituted alkenes leads to efficient formation of both dimeric and cross [2+2]-cycloaddition products. Qualitatively similar observations were made in a study of the photochemistry of N-trimethylsilylmethyl-2-pyridone. The combined results demonstrate that [2+2]-photocycloaddition is a more efficient excited state reaction pathway for the uracil and pyridone substrates as compared to other processes, such as ylide-forming trimethylsilyl group C-to-O migration. Finally, photoreactions of N-trimethylsilylmethyl-2-pyrrolidone in solutions containing dipolarophiles, such as methyl acrylate, lead to the formation of the desilylation product, N-methyl-2-pyrrolidone by way of a simple, non-ylide generating, protodesilylation process. In addition, observations were made which show that orbital symmetry allowed photocycloreversion reactions of dimeric uracil derivatives, involving cyclobutane ring splitting, to take place. These processes, which lead to the formation of monomeric uracils, appear to be stimulated by the presence of electron donor groups on the cyclobutane ring, a likely result of a new SET promoted cyclobutane ring cleavage pathway. In the cases of N-trimethylsilylmethyl-substituted cyclobutane derivatives that possess phthalimide groups, highly efficient excited state cleavage of the cyclobutane moiety occurs to produce uracil derivatives and corresponding vinyl phthalimide.

Divergence of biochemical function in the HAD superfamily: D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB).

D-Glycero-d-manno-heptose-1,7-bisphosphate phosphatase (GmhB) is a member of the histidinol-phosphate phosphatase (HisB) subfamily of the haloalkanoic acid dehalogenase (HAD) enzyme superfamily. GmhB supports two divergent biochemical pathways in bacteria: the d-glycero-d-manno-heptose-1alpha-GDP pathway (in S-layer glycoprotein biosynthesis) and the l-glycero-d-manno-heptose-1beta-ADP pathway (in lipid A biosynthesis). Herein, we report the comparative analysis of substrate recognition in selected GmhB orthologs. The substrate specificity of the l-glycero-d-manno-heptose-1beta-ADP pathway GmhB from Escherichia coli K-12 was evaluated using hexose and heptose bisphosphates, histidinol phosphate, and common organophosphate metabolites. Only d-glycero-d-manno-heptose 1beta,7-bisphosphate (k(cat)/K(m) = 7 x 10(6) M(-1) s(-1)) and d-glycero-d-manno-heptose 1alpha,7-bisphosphate (k(cat)/K(m) = 7 x 10(4) M(-1) s(-1)) displayed physiologically significant substrate activity. (31)P NMR analysis demonstrated that E. coli GmhB selectively removes the C(7) phosphate. Steady-state kinetic inhibition studies showed that d-glycero-d-manno-heptose 1beta-phosphate (K(is) = 60 microM, and K(ii) = 150 microM) and histidinol phosphate (K(is) = 1 mM, and K(ii) = 6 mM), while not hydrolyzed, do in fact bind to E. coli GmhB, which leads to the conclusion that nonproductive binding contributes to substrate discrimination. High catalytic efficiency and a narrow substrate range are characteristic of a well-evolved metabolic enzyme, and as such, E. coli GmhB is set apart from most HAD phosphatases (which are typically inefficient and promiscuous). The specialization of the biochemical function of GmhB was examined by measuring the kinetic constants for hydrolysis of the alpha- and beta-anomers of d-glycero-d-manno-heptose 1beta,7-bisphosphate catalyzed by the GmhB orthologs of the l-glycero-d-manno-heptose 1beta-ADP pathways operative in Bordetella bronchiseptica and Mesorhizobium loti and by the GmhB of the d-glycero-d-manno-heptose 1alpha-GDP pathway operative in Bacteroides thetaiotaomicron. The results show that although each of these representatives possesses physiologically significant catalytic activity toward both anomers, each displays substantial anomeric specificity. Like E. coli GmhB, B. bronchiseptica GmhB and M. loti GmhB prefer the beta-anomer, whereas B. thetaiotaomicron GmhB is selective for the alpha-anomer. By determining the anomeric configuration of the physiological substrate (d-glycero-d-manno-heptose 1,7-bisphosphate) for each of the four GmhB orthologs, we discovered that the anomeric specificity of GmhB correlates with that of the pathway kinase. The conclusion drawn from this finding is that the evolution of the ancestor to GmhB in the HisB subfamily provided for specialization toward two distinct biochemical functions.

Nature and kinetic analysis of carbon-carbon bond fragmentation reactions of cation radicals derived from SET-oxidation of lignin model compounds.

Features of the oxidative cleavage reactions of diastereomers of dimeric lignin model compounds, which are models of the major types of structural units found in the lignin backbone, were examined. Cation radicals of these substances were generated by using SET-sensitized photochemical and Ce(IV) and lignin peroxidase promoted oxidative processes, and the nature and kinetics of their C-C bond cleavage reactions were determined. The results show that significant differences exist between the rates of cation radical C1-C2 bond cleavage reactions of 1,2-diaryl-(ß-1) and 1-aryl-2-aryloxy-(ß-O-4) propan-1,3-diol structural units found in lignins. Specifically, under all conditions C1-C2 bond cleavage reactions of cation radicals of the ß-1 models take place more rapidly than those of the ß-O-4 counterparts. The results of DFT calculations on cation radicals of the model compounds show that the C1-C2 bond dissociation energies of the ß-1 lignin model compounds are significantly lower than those of the ß-O-4 models, providing clear evidence for the source of the rate differences.

Human symbiont Bacteroides thetaiotaomicron synthesizes 2-keto-3-deoxy-D-glycero-D- galacto-nononic acid (KDN).

The proper functioning of the human intestine is dependent on its bacterial symbionts, the most predominant of which belong to the Phylum Bacteroidetes. These bacteria are known to use variable displays of multiple capsular polysaccharides (CPs) to aid in their survival and foraging within the intestine. Bacteroides thetaiotaomicron is a prominent human gut symbiont and a remarkably versatile glycophile. The structure determination of the CPs, encoded by the eight CP loci, is the key to understanding the mechanism of this organism's adaptation on a molecular level. Herein, we report the bioinformatics-based discovery and chemical demonstration of a biosynthetic pathway that forms and cytidylates 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), most likely for inclusion in the CP encoded by B. thetaiotaomicron CP locus 7.

Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia.

Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of l-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in Escherichia coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an insilico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept l-arginine, l-arginine ethyl ester, N(alpha)-benzoyl-l-arginine, and N(omega)-amino-l-arginine as substrates but not agmatine, l-homoarginine, N(alpha)-benzoyl-l-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424-alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation (k(1)=80s(-1)) and hydrolysis (k(2)=35s(-1)) of the intermediate were determined. The comparatively lower value of the steady-state rate constant (k(cat)=2.6s(-1)), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-Nitroso-l-homocysteine was shown to be an active site directed, irreversible inhibitor whereas N(omega)-cyano-l-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.

Single electron transfer-promoted photocyclization reactions of linked acceptor-polydonor systems: effects of chain length and type on the efficiencies of macrocyclic ring-forming photoreactions of tethered alpha-silyl ether phthalimide substrates.

Results of an investigation, aimed at gaining information about the factors governing the efficiencies of single electron transfer (SET)-promoted photocyclization reactions of linked acceptor-polydonor systems, are described. One set of substrates used in this effort includes alpha-trimethylsilyl ether terminated, polymethylene- and polyethylenoxy-tethered phthalimides and 2,3-naphthalimides. Photocyclization reactions of the polyethylenoxy-linked phthalimides and naphthalimides were observed to take place in higher chemical yields and with larger quantum efficiencies than those of analogs containing polymethylene tethers of near equal length. These findings show that the rates of formation of 1,omega-zwitterionic biradicals that serve as key intermediates in the photocyclization processes are enhanced in substances that contain oxygen donor sites in the chain. The findings suggest that these donor sites facilitate both initial SET to acceptor excited states and ensuing intrachain SET, resulting in migration of the cation radical center to the terminal alpha-trimethylsilyl ether position. In addition, an inverse relationship was observed between the quantum yields of photocyclization reactions of the tethered phthalimides and naphthalimides and the length of the polyethylenoxy chain. Finally, the roles played by chain type and length in governing photoreaction efficiencies were investigated by using intramolecular competition in photoreactions of polyethylenoxy and polymethylene bis-tethered phthalimides. Mechanistic interpretations and synthetic consequences of the observations made in this study are discussed.