Reversibility and efficiency in electrocatalytic energy conversion and lessons from enzymes

Enzymes are long established as extremely efficient catalysts. Here, we show that enzymes can also be extremely efficient electrocatalysts (catalysts of redox reactions at electrodes). Despite being large and electronically insulating through most of their volume, some enzymes, when attached to an electrode, catalyze electrochemical reactions that are otherwise extremely sluggish (even with the best synthetic catalysts) and require a large overpotential to achieve a useful rate. These enzymes produce high electrocatalytic currents, displayed in single bidirectional voltammetric waves that switch direction (between oxidation and reduction) sharply at the equilibrium potential for the substrate redox couple. Notoriously irreversible processes such as CO(2) reduction are thereby rendered electrochemically reversible--a consequence of molecular evolution responding to stringent biological drivers for thermodynamic efficiency. Enzymes thus set high standards for the catalysts of future energy technologies.     

Substrate activation for O2 reactions by oxidized metal centers in biology

The uncatalyzed reactions of O₂ (S = 1) with organic substrates (S = 0) are thermodynamically favorable but kinetically slow because they are spin-forbidden and the one-electron reduction potential of O₂ is unfavorable. In nature, many of these important O₂ reactions are catalyzed by metalloenzymes. In the case of mononuclear non-heme iron enzymes, either Fe^II or Fe^III can play the catalytic role in these spin-forbidden reactions. Whereas the ferrous enzymes activate O₂ directly for reaction, the ferric enzymes activate the substrate for O₂ attack. The enzyme–substrate complex of the ferric intradiol dioxygenases exhibits a low-energy catecholate to Fe^III charge transfer transition that provides a mechanism by which both the Fe center and the catecholic substrate are activated for the reaction with O₂. In this Perspective, we evaluate how the coupling between this experimentally observed charge transfer and the change in geometry and ligand field of the oxidized metal center along the reaction coordinate can overcome the spin-forbidden nature of the O₂ reaction.     

Air-stable, storable, and highly efficient chiral zirconium catalysts for enantioselective Mannich-type, aza Diels-Alder, aldol, and hetero Diels-Alder reactions

For the synthesis of optically active compounds, chiral catalysts have attracted much attention because large quantities of optically active molecules can be prepared from a small amount of a chiral source. However, many chiral catalysts are often unstable in air (oxygen) and/or in the presence of water. This is especially the case in chiral Lewis acid catalysis, because most Lewis acids are air- and moisture-sensitive. Therefore, many catalysts are prepared in situ in an appropriate solvent just before use, and they cannot be stored for extended periods. We have developed air-stable, storable, and highly efficient chiral zirconium Lewis acids. The catalysts promoted asymmetric Mannich-type, aza Diels-Alder, aldol, and hetero Diels-Alder reactions efficiently with high enantioselectivities. A key to stabilizing the catalysts is an appropriate combination of chiral zirconium Lewis acids with molecular sieves, and the zirconium-molecular sieves-combined catalysts can be stored for extended periods in air at room temperature without loss of activity. Moreover, it has been demonstrated that the catalysts can be recovered and reused.     

Cobalt-dithiolene complexes for the photocatalytic and electrocatalytic reduction of protons in aqueous solutions

Artificial photosynthesis (AP) is a promising method of converting solar energy into fuel (H(2)). Harnessing solar energy to generate H(2) from H(+) is a crucial process in systems for artificial photosynthesis. Widespread application of a device for AP would rely on the use of platinum-free catalysts due to the scarcity of noble metals. Here we report a series of cobalt dithiolene complexes that are exceptionally active for the catalytic reduction of protons in aqueous solvent mixtures. All catalysts perform visible-light-driven reduction of protons from water when paired with as the photosensitizer and ascorbic acid as the sacrificial donor. Photocatalysts with electron withdrawing groups exhibit the highest activity with turnovers up to 9,000 with respect to catalyst. The same complexes are also active electrocatalysts in 11 acetonitrile/water. The electrocatalytic mechanism is proposed to be ECEC, where the Co dithiolene catalysts undergo rapid protonation once they are reduced to . Subsequent reduction and reaction with H(+) lead to H(2) formation. Cobalt dithiolene complexes thus represent a new group of active catalysts for the reduction of protons.     

The biology and pathology of oxygen radicals.

Superoxide radicals (O2-) are commonplace products of the biological reduction of oxygen. Their intrinsic reactivity and ability to generate other more reactive entities constitute a threat to cellular integrity. Superoxide dismutases, enzymes that catalytically scavenge these radicals, have evolved to meet this threat. These metalloenzymes are essential for respiring organisms to survive. Several compounds, such as the antibiotic streptonigrin and the herbicide paraquat, augment the production rate of O2- inside cells. This accounts for the oxygen-enhancement of their lethality. Some bacteria respond to this artificially increased rate of O2- production by synthesizing additional superoxide dismutase. Ionizing radiation generates O2- in its passage through oxygenated aqueous media, and superoxide dismutase added to the suspending medium, decreases the oxygen-enhancement of the lethality of such irradiation of the bacterium Escherichia coli. Production of O2- by activated neutrophils is clinically significant, since it is an important component of the bactericidal actions of these cells and the inflammatory process. Superoxide dismutases exert an anti-inflammatory action that may be useful in managing inflammations.     

High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay.

A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites.     

Structure and metal-dependent mechanism of peptidoglycan deacetylase, a streptococcal virulence factor

Streptococcus pneumoniae peptidoglycan GlcNAc deacetylase (SpPgdA) protects the Gram-positive bacterial cell wall from host lysozymes by deacetylating peptidoglycan GlcNAc residues. Deletion of the pgda gene has been shown to result in hypersensitivity to lysozyme and reduction of infectivity in a mouse model. SpPgdA is a member of the family 4 carbohydrate esterases, for which little structural information exists, and no catalytic mechanism has yet been defined. Here we describe the native crystal structure and product complexes of SpPgdA biochemical characterization and mutagenesis. The structural data show that SpPgdA is an elongated three-domain protein in the crystal. The structure, in combination with mutagenesis, shows that SpPgdA is a metalloenzyme using a His-His-Asp zinc-binding triad with a nearby aspartic acid and histidine acting as the catalytic base and acid, respectively, somewhat similar to other zinc deacetylases such as LpxC. The enzyme is able to accept GlcNAc(3) as a substrate (K(m) = 3.8 mM, k(cat) = 0.55 s(-1)), with the N-acetyl of the middle sugar being removed by the enzyme. The data described here show that SpPgdA and the other family 4 carbohydrate esterases are metalloenzymes and present a step toward identification of mechanism-based inhibitors for this important class of enzymes.     

Controlling the enantioselectivity of enzymes by directed evolution: practical and theoretical ramifications

A fundamentally new approach to asymmetric catalysis in organic chemistry is described based on the in vitro evolution of enantioselective enzymes. It comprises the appropriate combination of gene mutagenesis and expression coupled with an efficient high-throughput screening system for evaluating enantioselectivity (enantiomeric excess assay). Several such cycles lead to a "Darwinistic" process, which is independent of any knowledge concerning the structure or the mechanism of the enzyme being evolved. The challenge is to choose the optimal mutagenesis methods to navigate efficiently in protein sequence space. As a first example, the combination of error-prone mutagenesis, saturation mutagenesis, and DNA-shuffling led to a dramatic enhancement of enantioselectivity of a lipase acting as a catalyst in the kinetic resolution of a chiral ester. Mutations at positions remote from the catalytically active center were identified, a surprising finding, which was explained on the basis of a novel relay mechanism. The scope and limitations of the method are discussed, including the prospect of directed evolution of stereoselective hybrid catalysts composed of robust protein hosts in which transition metal centers have been implanted.     

Occurrence of short-sized oligo(A) fragments during course of cell cycle and ageing

Affinity chromatography of nucleic acids precipitated by N-cetyl-N,N,N-trimethyl ammonium bromide on poly(U)-Sepharose has proved to be a suitable method for a nearly quantitative isolation of oligo(A) sequences down to a chain length of 4 nucleotide units. Analysis of short oligo(A) fragments in synchronized L5178y mouse lymphoma cells after labeling with [3H]Ado revealed that the percentage of A2-6 sequences on the total radioactivity amounted in S-phase cells to 1.6%, while the value obtained for the stationary L-cell system was 8.0%. The alterations of occurrence and chain length distribution of short oligo(A) fragments during ageing were studied in two age groups of female quails: mature (250-320 days old) and senescent animals (3-3.5 yr old). It was found that the amount of low molecular weight oligo(A) fragments gradually decreases during ageing of the animals; the amount in the mature animal group was significantly higher (6-fold) than in the old animal group. The decreased amounts of oligo(A) during S phase and ageing could in part be due to posttranslational modification of enzymes involved in poly(A) metabolism. It could be demonstrated that both homogeneous poly(A) anabolic poly(A) polymerase and homogeneous poly(A) catabolic endoribonuclease IV are phosphorylated by nuclear protein kinase NI.     

Metalloenzyme-like catalyzed isomerizations of sugars by Lewis acid zeolites

Isomerization of sugars is used in a variety of industrially relevant processes and in glycolysis. Here, we show that hydrophobic zeolite beta with framework tin or titanium Lewis acid centers isomerizes sugars, e.g., glucose, via reaction pathways that are analogous to those of metalloenzymes. Specifically, experimental and theoretical investigations reveal that glucose partitions into the zeolite in the pyranose form, ring opens to the acyclic form in the presence of the Lewis acid center, isomerizes into the acyclic form of fructose, and finally ring closes to yield the furanose product. The zeolite catalysts provide processing advantages over metalloenzymes such as an ability to work at higher temperatures and in acidic conditions that allow for the isomerization reaction to be coupled with other important conversions.     

Modification of bacterial artificial chromosomes through Chi-stimulated homologous recombination and its application in zebrafish transgenesis

The modification of yeast artificial chromosomes through homologous recombination has become a useful genetic tool for studying gene function and enhancer/promoter activity. However, it is difficult to purify intact yeast artificial chromosome DNA at a concentration sufficient for many applications. Bacterial artificial chromosomes (BACs) are vectors that can accommodate large DNA fragments and can easily be purified as plasmid DNA. We report herein a simple procedure for modifying BACs through homologous recombination using a targeting construct containing properly situated Chi sites. To demonstrate a usage for this technique, we modified BAC clones containing the zebrafish GATA-2 genomic locus by replacing the first coding exon with the green fluorescent protein (GFP) reporter gene. Molecular analyses confirmed that the modification occurred without additional deletions or rearrangements of the BACs. Microinjection demonstrated that GATA-2 expression patterns can be recapitulated in living zebrafish embryos by using these GFP-modified GATA-2 BACs. Embryos microinjected with the modified BAC clones were less mosaic and had improved GFP expression in hematopoietic progenitor cells compared with smaller plasmid constructs. The precise modification of BACs through Chi-stimulated homologous recombination should be useful for studying gene function and regulation in cultured cells or organisms where gene transfer is applicable.     

Characterization of hydrogen-uptake activity in the hyperthermophile Pyrodictium brockii.

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105 degrees C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have characterized P. brockii hydrogen-uptake activity with regard to temperature, ability to couple hydrogen oxidation to artificial electron acceptor reduction, sensitivity to O2, and cellular localization. The hydrogen-uptake activity was localized predominantly in a particulate fraction, was reversibly inhibited by O2, and coupled H2 uptake to the reduction of positive potential artificial electron acceptors. Comparisons between these results and those of the well-studied hydrogen-uptake hydrogenase from the mesophile Bradyrhizobium japonicum showed the two enzymes to be similar despite the very different natural environments of the organisms. However, the optimum temperature for activity differed greatly in the two organisms. We have also used immunological and genetic probes specific to the 65-kDa subunit of B. japonicum hydrogenase to assay crude extracts and genomic DNA, respectively, from P. brockii and found the enzymes to be similar in these respects as well. In addition, we report a formulation for artificial seawater capable of sustaining the growth of P. brockii.     

Pertussis toxin-sensitive G proteins are transported toward synaptic terminals by fast axonal transport.

We find that half of the pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in the squid (Loligo pealei) giant axon is cytoplasmic and that this species of G protein is intermediate in size between the two forms present in axolemma. This G protein is transported toward synaptic terminals at 44 mm/day. Moreover, these data are consistent with there being two additional steps leading to the maturation of G proteins: (i) association with and transport on intracellular organelles and (ii) modification at the time of transfer to the plasmalemma resulting in a molecular weight shift. Since the other two components of G protein-mediated signal transduction pathways, receptors and effector enzymes, are known to be delivered to the synaptic terminals by fast axonal transport, our findings introduce the possibility that these three macromolecules are assembled as a complex in the cell body and delivered together to the plasma membrane of the axon and synaptic terminals.     

Chiral stereoisomeric molecules in the treatment of arthritis.

Pharmacokinetic and pharmacodynamic properties of drugs and their ultimate therapeutic effects are often significantly influenced by interactions between the geometry of host receptors, host enzymes, and the three-dimensional structure of drugs. Drug molecules that are mirror images of each other are chiral stereoisomers, and such chiral isomer compounds are commonly used as therapeutic agents by rheumatologists either as racemates (mixtures of chiral isomers) or as pure stereoisomers. Understanding and using such stereoisomeric drugs may lead to lower risks of drug toxicity, better therapeutic indices, and newer approaches for the treatment of articular disorders. A review of the properties of these special isomers is presented, and their therapeutic advantages are discussed.     

Protein modification in aging

The age-related accumulation of abnormal forms of enzymes is attributable to posttranslational modification of protein structure and to a progressive loss with age of proteases that preferentially degrade the modified forms. The protein modifications include, but are not limited to: the oxidation of amino acid side chains (especially, side chains of prolyl, arginyl, lysyl and histidinyl residues) by mixed-function oxidation systems; the deamidation of asparaginyl and glutaminyl residues; the racemization and isomerization of aspartyl and asparaginyl residues; the isomerization of prolyl residues; the oxidation of cysteine sulfhydryl groups; and spontaneous changes in protein conformation that are apparently unlinked to changes in amino acid composition. Evidence supporting the roles of these protein modifications and of the proteases that degrade abnormal enzymes during aging is discussed, as well as a consideration of some technical limitations of the methods used in their study.     

Catalytic enantioselective intermolecular cycloadditions of 2-diazo-3,6-diketoester-derived carbonyl ylides with alkene dipolarophiles

Catalyzed cascade reactions that generate molecular complexity rapidly and in an enantioselective manner are attractive methods for asymmetric synthesis. In the present article, chiral rhodium catalysts are shown to effect such a transformation by using a range of 2-diazo-3,6-diketoesters with bicyclo[2.2.1]alkenes and styrenes as reaction partners. The reactions are likely to proceed by formation of a catalyst-complexed carbonyl ylide from the diazo compound, followed by intermolecular cycloaddition with the alkene dipolarophile. It was possible to obtain high levels of asymmetric induction [up to 89% enantiomeric excess (ee) and 92% ee for the two chiral catalysts investigated]. Enantioselectivity is not highly sensitive to substituent variation at the ketone that forms the ylide; however, branching does improve ee. Observations of dipolarophile-dependent enantiofacial selectivity in the cycloadditions indicate that the dipolarophile can be intimately involved in the enantiodiscrimination process.     

Molecular and functional analysis of nicotinate catabolism in Eubacterium barkeri

The anaerobic soil bacterium Eubacterium barkeri catabolizes nicotinate to pyruvate and propionate via a unique fermentation. A full molecular characterization of nicotinate fermentation in this organism was accomplished by the following results: (i) A 23.2-kb DNA segment with a gene cluster encoding all nine enzymes was cloned and sequenced, (ii) two chiral intermediates were discovered, and (iii) three enzymes were found, completing the hitherto unknown part of the pathway. Nicotinate dehydrogenase, a (nonselenocysteine) selenium-containing four-subunit enzyme, is encoded by ndhF (FAD subunit), ndhS (2 x [2Fe-2S] subunit), and by the ndhL/ndhM genes. In contrast to all enzymes of the xanthine dehydrogenase family, the latter two encode a two-subunit molybdopterin protein. The 6-hydroxynicotinate reductase, catalyzing reduction of 6-hydroxynicotinate to 1,4,5,6-tetrahydro-6-oxonicotinate, was purified and shown to contain a covalently bound flavin cofactor, one [2Fe-2S](2+/1+) and two [4Fe-4S](2+/1+) clusters. Enamidase, a bifunctional Fe-Zn enzyme belonging to the amidohydrolase family, mediates hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to ammonia and (S)-2-formylglutarate. NADH-dependent reduction of the latter to (S)-2-(hydroxymethyl)glutarate is catalyzed by a member of the 3-hydroxyisobutyrate/phosphogluconate dehydrogenase family. A [4Fe-4S]-containing serine dehydratase-like enzyme is predicted to form 2-methyleneglutarate. After the action of the coenzyme B(12)-dependent 2-methyleneglutarate mutase and 3-methylitaconate isomerase, an aconitase and isocitrate lyase family pair of enzymes, (2R,3S)-dimethylmalate dehydratase and lyase, completes the pathway. Genes corresponding to the first three enzymes of the E. barkeri nicotinate catabolism were identified in nine Proteobacteria.     

O-GlcNAcylation: a novel post-translational mechanism to alter vascular cellular signaling in health and disease: focus on hypertension

O-Linked attachment of β-N-acetyl-glucosamine (O-GlcNAc) on serine and threonine residues of nuclear and cytoplasmic proteins is a highly dynamic posttranslational modification that plays a key role in signal transduction pathways. Preliminary data show that O-GlcNAcylation may represent a key regulatory mechanism in the vasculature, modulating contractile and relaxant responses. Proteins with an important role in vascular function, such as endothelial nitric oxide synthase, sarcoplasmic reticulum Ca²⁺-ATPase, protein kinase C, mitogen-activated protein kinases, and proteins involved in cytoskeleton regulation and microtubule assembly are targets for O-GlcNAcylation, indicating that this posttranslational modification may play an important role in vascular reactivity. Here, we will focus on a few specific pathways that contribute to vascular function and cardiovascular disease–associated vascular dysfunction, and the implications of their modification by O-GlcNAc. New chemical tools have been developed to detect and study O-GlcNAcylation, including inhibitors of O-GlcNAc enzymes, chemoenzymatic tagging methods, and quantitative proteomics strategies; these will also be briefly addressed. An exciting challenge in the future will be to better understand the cellular dynamics of this posttranslational modification, as well as the signaling pathways and mechanisms by which O-GlcNAc is regulated on specific proteins in the vasculature in health and disease.     

Spatio-temporal correlations can drastically change the response of a MAPK pathway

Multisite covalent modification of proteins is omnipresent in eukaryotic cells. A well-known example is the mitogen-activated protein kinase (MAPK) cascade where, in each layer of the cascade, a protein is phosphorylated at two sites. It has long been known that the response of a MAPK pathway strongly depends on whether the enzymes that modify the protein act processively or distributively. A distributive mechanism, in which the enzyme molecules have to release the substrate molecules in between the modification of the two sites, can generate an ultrasensitive response and lead to hysteresis and bistability. We study by Green’s Function Reaction Dynamics (GFRD), a stochastic scheme that makes it possible to simulate biochemical networks at the particle level in time and space, a dual phosphorylation cycle in which the enzymes act according to a distributive mechanism. We find that the response of this network can differ dramatically from that predicted by a mean-field analysis based on the chemical rate equations. In particular, rapid rebindings of the enzyme molecules to the substrate molecules after modification of the first site can markedly speed up the response and lead to loss of ultrasensitivity and bistability. In essence, rapid enzyme-substrate rebindings can turn a distributive mechanism into a processive mechanism. We argue that slow ADP release by the enzymes can protect the system against these rapid rebindings, thus enabling ultrasensitivity and bistability.