Adenosylcobalamin-dependent isomerases: new insights into structure and mechanism

Adenosylcobalamin-dependent isomerases catalyze a variety of chemically difficult 1,2-rearrangements that proceed through a mechanism involving free radical intermediates. These radicals are initially generated by homolysis of the cobalt-carbon bond of the coenzyme. Recently, the crystal structures of several of these enzymes have been solved, revealing two modes of coenzyme binding and highlighting the role of the protein in controlling the rearrangement of reactive substrate radical intermediates. Complementary data from kinetic, spectroscopic and theoretical studies have produced insights into the mechanism by which substrate radicals are generated at the active site, and the pathways by which they rearrange.     

Free radical mechanisms in enzyme reactions

Free radicals are formed in prosthetic groups or amino acid residues of certain enzymes. These free radicals are closely related to the activation process in enzyme catalysis, but their formation does not always result in the formation of substrate free radicals as a product of the enzyme reactions. The role of free radicals in enzyme catalysis is discussed.     

Substrate specificities and excision kinetics of DNA glycosylases involved in base-excision repair of oxidative DNA damage

Reactive oxygen-derived species such as free radicals are formed in living cells by normal metabolism and exogenous sources, and cause a variety of types of DNA damage such as base and sugar damage, strand breaks and DNA-protein cross-links. Living organisms possess repair systems that repair DNA damage. Oxidative DNA damage caused by free radicals and other oxidizing agents is mainly repaired by base-excision repair (BER), which involves DNA glycosylases in the first step of the repair process. These enzymes remove modified bases from DNA by hydrolyzing the glycosidic bond between the modified base and the sugar moiety, generating an apurinic/apyrimidinic (AP) site. Some also possess AP lyase activity that subsequently cleaves DNA at AP sites. Many DNA glycosylases have been discovered and isolated, and their reaction mechanisms and substrate specificities have been elucidated. Most of the known products of oxidative damage to DNA are substrates of DNA glycosylases with broad or narrow substrate specificities. Some possess cross-activity and remove both pyrimidine- and purine-derived lesions. Overlapping activities between enzymes also exist. Studies of substrate specificities have been performed using either oligodeoxynucleotides with a single modified base embedded at a specific position or damaged DNA substrates containing a multiplicity of pyrimidine- and purine-derived lesions. This paper reviews the substrate specificities and excision kinetics of DNA glycosylases that have been investigated with the use of gas chromatography/mass spectrometry and DNA substrates with multiple lesions.     

Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay)

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.     

The oxygen effect in radiation inactivation of DNA and enzymes

A survey is made of literature data dealing with the influence of oxygen on radiation effects in biologically active DNA and enzymes irradiated extracellularly. There is evidence that oxygen takes part in physico-chemical events, directly or indirectly produced by radiation in several ways: from scavenging reducing primary water radicals to reacting directly with macromolecular radical sites. There is evidence that radiation-induced secondary radicals, originating from a variety of low molecular weight biomolecules, can react with DNA and enzymes in their native state, and produce inactivation. By reaction with oxygen secondary radicals become peroxidized and in this form are generally more harmful to biological macromolecules. There are indications that thiol peroxy radicals can also act in the same way. Possible implications for the oxygen effect observed in vivo are discussed.     

Free radicals in physiology and pathology.

Free radicals are molecules with odd number of electrons and a high instability. Free radicals, which can occur in both organic (i.e., quinones) and inorganic molecules (i.e., O2-), are very reactive and their reactions are critical for the normal activity of a wide spectrum of biologic processes. They are also produced in the catalytic action of a variety of cellular enzymes and electron transport processes and are implicated in a number of physiologic and pathologic processes. Organisms can be exposed to free radicals in many ways other than through the processes of normal metabolism. Irradiation of organisms with electromagnetic radiation generates primary radicals (e-aq, OH., and H.), which can then undergo secondary reactions with dissolved O2 or with cellular solutes. In addition, a wide variety of environmental agents (drugs capable of redox cycling, and xenobiotics that can form free radical metabolites) including the aging process cause free radical damage to cells. This review deals with the reactions they can undergo and discusses the free radicals related to toxicology.     

Effects of site-directed mutations in Escherichia coli succinate dehydrogenase on the enzyme activity and production of superoxide radicals.

Escherichia coli succinate dehydrogenase (SdhCDAB) catalyzes the oxidation of succinate to fumarate in the Krebs cycle, and during turnover, it produces superoxide radicals. SdhCDAB is a good model system for the succinate dehydrogenase (Sdh) found in the mitochondrial respiratory chain (complex II), as the subunits are structural homologues. Although mutations in sdh genes are reportedly associated with a variety of mitochondria-related diseases, the molecular mechanism of these diseases is poorly understood. We have investigated the effects of site-directed mutations around the heme (SdhD-H71L and SdhC-H91L), and at the ubiquinone-binding site (Q site; SdhC-I28E), on enzyme activity and production of superoxide radicals. The mutations SdhD-H71L and SdhC-I28E, but not SdhC-H91L, significantly reduce the succinate-ubiquinone reductase activity of the enzyme. All 3 mutant enzymes produce more superoxide than the wild-type enzyme, indicating that disturbance of the heme or the Q site can enhance superoxide production. The presence of a Q-site inhibitor reduces superoxide production significantly. Furthermore, the yield of superoxide is substrate dependent and increases with succinate concentration from 0.1 to 10 mmol/L. Our results indicate that, in SdhCDAB, the Q site with bound ubiquinone is an important source of superoxide radicals.     

Mammalian alcohol dehydrogenase — Functional and structural implications

Mammalian alcohol dehydrogenase (ADH) constitutes a complex system with different forms and extensive multiplicity (ADH1–ADH6) that catalyze the oxidation and reduction of a wide variety of alcohols and aldehydes. The ADH1 enzymes, the classical liver forms, are involved in several metabolic pathways beside the oxidation of ethanol, e.g. norepinephrine, dopamine, serotonin and bile acid metabolism. This class is also able to further oxidize aldehydes into the corresponding carboxylic acids, i.e. dismutation. ADH2, can be divided into two subgroups, one group consisting of the human enzyme together with a rabbit form and another consisting of the rodent forms. The rodent enzymes almost lack ethanol-oxidizing capacity in contrast to the human form, indicating that rodents are poor model systems for human ethanol metabolism. ADH3 (identical to glutathione-dependent formaldehyde dehydrogenase) is clearly the ancestral ADH form and S-hydroxymethylglutathione is the main physiological substrate, but the enzyme can still oxidize ethanol at high concentrations. ADH4 is solely extrahepatically expressed and is probably involved in first pass metabolism of ethanol beside its role in retinol metabolism. The higher classes, ADH5 and ADH6, have been poorly investigated and their substrate repertoire is unknown. The entire ADH system can be seen as a general detoxifying system for alcohols and aldehydes without generating toxic radicals in contrast to the cytochrome P450 system.     

Adenosylcobalamin enzymes: theory and experiment begin to converge

Adenosylcobalamin (coenzyme B(12)) serves as the cofactor for a group of enzymes that catalyze unusual rearrangement or elimination reactions. The role of the cofactor as the initiator of reactive free radicals needed for these reactions is well established. Less clear is how these enzymes activate the coenzyme towards homolysis and control the radicals once generated. The availability of high resolution X-ray structures combined with detailed kinetic and spectroscopic analyses have allowed several adenosylcobalamin enzymes to be computationally modeled in some detail. Computer simulations have generally obtained good agreement with experimental data and provided valuable insight into the mechanisms of these unusual reactions. Importantly, atomistic modeling of the enzymes has allowed the role of specific interactions between protein, substrate and coenzyme to be explored, leading to mechanistic predictions that can now be tested experimentally. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

The radical chemistry of galactose oxidase

Galactose oxidase is a free radical metalloenzyme containing a novel metalloradical complex, comprised of a protein radical coordinated to a copper ion in the active site. The unusually stable protein radical is formed from the redox-active side chain of a cross-linked tyrosine residue (Tyr-Cys). Biochemical studies on galactose oxidase have revealed a new class of oxidation mechanisms based on this free radical coupled-copper catalytic motif, defining an emerging family of enzymes, the radical-copper oxidases. Isotope kinetics and substrate reaction profiling have provided insight into the elementary steps of substrate oxidation in these enzymes, complementing structural studies on their active site. Galactose oxidase is remarkable in the extent to which free radicals are involved in all aspects of the enzyme function: serving as a key feature of the active site structure, defining the characteristic reactivity of the complex, and directing the biogenesis of the Tyr-Cys cofactor during protein maturation.     

Controlling reaction specificity in pyridoxal phosphate enzymes

Pyridoxal 5'-phosphate enzymes are ubiquitous in the nitrogen metabolism of all organisms. They catalyze a wide variety of reactions including racemization, transamination, decarboxylation, elimination, retro-aldol cleavage, Claisen condensation, and others on substrates containing an amino group, most commonly α-amino acids. The wide variety of reactions catalyzed by PLP enzymes is enabled by the ability of the covalent aldimine intermediate formed between substrate and PLP to stabilize carbanionic intermediates at Cα of the substrate. This review attempts to summarize the mechanisms by which reaction specificity can be achieved in PLP enzymes by focusing on three aspects of these reactions: stereoelectronic effects, protonation state of the external aldimine intermediate, and interaction of the carbanionic intermediate with the protein side chains present in the active site. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Laccase versus Laccase-Like Multi-Copper Oxidase: A Comparative Study of Similar Enzymes with Diverse Substrate Spectra

Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyse the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. Owing to their broad substrate range, copper-containing laccases are versatile biocatalysts, capable of oxidizing numerous natural and non-natural industry-relevant compounds, with water as the sole by-product. In the present study, 10 of the 11 multi-copper oxidases, hitherto considered to be laccases, from fungi, plant and bacterial origin were compared. A substrate screen of 91 natural and non-natural compounds was recorded and revealed a fairly broad but distinctive substrate spectrum amongst the enzymes. Even though the enzymes share conserved active site residues we found that the substrate ranges of the individual enzymes varied considerably. The EC classification is based on the type of chemical reaction performed and the actual name of the enzyme often refers to the physiological substrate. However, for the enzymes studied in this work such classification is not feasible, even more so as their prime substrates or natural functions are mainly unknown. The classification of multi-copper oxidases assigned as laccases remains a challenge. For the sake of simplicity we propose to introduce the term “laccase-like multi-copper oxidase” (LMCO) in addition to the term laccase that we use exclusively for the enzyme originally identified from the sap of the lacquer tree Rhus vernicifera.     

Protection against oxygen radicals: an important defence mechanism studied in transgenic plants

Free radicals and other active derivatives of oxygen are inevitable by-products of biological redox reactions. Reduced oxygen species, such as hydrogen peroxide, the superoxide radical anion and hydroxyl radicals, inactivate enzymes and damage important cellular components. In addition, singlet oxygen, produced via formation of triplet state chlorophyll, is highly destructive. This oxygen species initiates lipid peroxidation, and produces lipid peroxy radicals and lipid hydroperoxides that are also very reactive. The increased production of toxic oxygen derivatives is considered to be a universal or common feature of stress conditions. Plants and other organisms have evolved a wide range of mechanisms to contend with this problem. The antioxidant defence system of the plant comprises a variety of antioxidant molecules and enzymes. Considerable interest has been focused on the ascorbate-glutathione cycle because it has a central role in protecting the chloroplasts and other cellular compartments from oxidative damage. It is clear that the capacity and activity of the antioxidative defence systems are important in limiting photo-oxidative damage and in destroying active oxygen species that are produced in excess of those normally required for signal transduction or metabolism. In our studies on this system, we became aware that the answers to many unresolved questions concerning the nature and regulation of the antioxidative defence system could not be obtained easily by either a purely physiological or purely biochemical approach. Transgenic plants offered us a means by which to achieve a more complete understanding of the roles of the enzymes involved in protection against stress of many types: environmental and man-made. The ability to engineer plants which express introduced genes at high levels provides an opportunity to manipulate the levels of these enzymes, and hence metabolism in vivo. Studies on transformed plants expressing increased activities of single enzymes of the antioxidative defence system indicate that it is possible to confer a degree of tolerence to stress by this means. However, attempts to increase stress resistance by simply increasing the activity of one of the antioxidant enzymes have not always been successful presumably because of the need for a balanced interaction of protective enzymes. The study of these transformed plants has allowed a more complete understanding of the roles of individual enzymes in metabolism. Protection against oxidative stress has become a feasible objective through the application of molecular genetic techniques in conjunction with a biochemical and physiological approach.     

Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.     

Structural studies of human alkyladenine glycosylase and E. coli 3-methyladenine glycosylase

Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine glycosylase (AlkA) are base excision repair glycosylases that recognize and excise a variety of alkylated bases from DNA. The crystal structures of these enzymes have provided insight into their substrate specificity and mechanisms of catalysis. Both enzymes utilize DNA bending and base-flipping mechanisms to expose and bind substrate bases. Crystal structures of AAG complexed to DNA suggest that the enzyme selects substrate bases through a combination of hydrogen bonding and the steric constraints of the active site, and that the enzyme activates a water molecule for an in-line backside attack of the N-glycosylic bond. In contrast to AAG, the structure of the AlkA-DNA complex suggests that AlkA substrate recognition and catalytic specificity are intimately integrated in a S(N)1 type mechanism in which the catalytic Asp238 directly promotes the release of modified bases.     

Role of the [2Fe-2S]2+ cluster in biotin synthase: mutagenesis of the atypical metal ligand arginine 260

Biotin synthase (BS) is an S-adenosylmethionine (AdoMet)-dependent radical enzyme that catalyzes the addition of sulfur to dethiobiotin. Like other AdoMet radical enzymes, BS contains a [4Fe-4S] cluster that is coordinated by a highly conserved CxxxCxxC sequence motif and by the methionyl amine and carboxylate of AdoMet. The close association of the [4Fe-4S]+ cluster with AdoMet facilitates reductive cleavage of the sulfonium and the generation of transient 5'-deoxyadenosyl radicals, which are then proposed to sequentially abstract hydrogen atoms from the substrate to produce carbon radicals at C9 and C6 of dethiobiotin. BS also contains a [2Fe-2S]2+ cluster located approximately 4-5 A from dethiobiotin, and we have proposed that a bridging sulfide of this cluster quenches the substrate radicals, leading to formation of the thiophane ring of biotin. In BS from Escherichia coli, the [2Fe-2S]2+ cluster is coordinated by cysteines 97, 128, and 188, and the atypical metal ligand, arginine 260. The evolutionary conservation of an arginine guanidinium as a metal ligand suggests a novel role for this residue in tuning the reactivity or stability of the [2Fe-2S]2+ cluster. In this work, we explore the effects of mutagenesis of Arg260 to Ala, Cys, His, and Met. Although perturbations in a number of characteristics of the [2Fe-2S]2+ cluster and the proteins are noted, the reconstituted enzymes have in vitro single-turnover activities that are 30-120% of that of the wild type. Further, in vivo expression of each mutant enzyme was sufficient to sustain growth of a bioB- mutant strain on dethiobiotin-supplemented medium, suggesting the enzymes were active and efficiently reconstituted by the in vivo iron-sulfur cluster (ISC) assembly system. Although we cannot exclude an as-yet-unidentified in vivo role in cluster repair or retention, we can conclude that Arg260 is not essential for the catalytic reaction of BS.     

A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes—Factors affecting enzymes,conversion and synergy

Lignocellulose is a complex substrate which requires a variety of enzymes, acting in synergy, for its complete hydrolysis. These synergistic interactions between different enzymes have been investigated in order to design optimal combinations and ratios of enzymes for different lignocellulosic substrates that have been subjected to different pretreatments. This review examines the enzymes required to degrade various components of lignocellulose and the impact of pretreatments on the lignocellulose components and the enzymes required for degradation. Many factors affect the enzymes and the optimisation of the hydrolysis process, such as enzyme ratios, substrate loadings, enzyme loadings, inhibitors, adsorption and surfactants. Consideration is also given to the calculation of degrees of synergy and yield. A model is further proposed for the optimisation of enzyme combinations based on a selection of individual or commercial enzyme mixtures. The main area for further study is the effect of and interaction between different hemicellulases on complex substrates.     

The localization of trehalase in polyacrylamide gels with eugenol by a coupled enzyme assay

Methods for the localization of trehalase (α, α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) activity following electrophoresis in nondenaturing polyacrylamide gels have been developed, in which trehalose hydrolysis is coupled to oxidation of the peroxidase substrate eugenol (2-methoxy-4-allyl phenol) by the use of glucose oxidase (EC 1.1.3.4), and peroxidase (EC 1.11.1.7) as ancillary enzymes. The basis for this procedure stems from the fact that free radicals of eugenol which are generated during the coupled trehalase assay condense to form a white precipitate whose location in the gel may be determined by densitometric scanning and whose surface area is a linear function of the enzyme level subjected to electrophoresis.     

Enzymes in non-aqueous solvents

For a variety of reasons including increased recognition of the large degree of association, by non-polar interaction, of enzymes with other cellular components such as membranes, enzymes are increasingly being investigated in mixed solvents less polar than water. Such solvents may be quite relevant because their polarity more nearly resembles the natural cellular microenvironment than does pure water. The single most important criterion in selecting a non-aqueous solvent is its compatibility with the maintenance of the enzyme's catalytic activity, which must be determined experimentally for each enzyme. Non-aqueous solvents have a variety of effects on enzymes: they may bind specifically, compete with substrate binding, dissociate multimers, shift an equilibrium between two enzyme conformations, alter the amount of helix, react with the enzyme, stabilize or destabilize the enzyme, and affect the rate of the catalytic reaction in several different ways. Typically, modest concentrations of hydroxylic solvents have little effect on rates, and may even enhance the rate significantly. Higher concentrations give lower rates, in a solvent-specific and enzyme-specific manner. Hydroxylic solvents may replace water as acceptor of a phosphoryl, glycosyl, or acyl group produced by a hydrolytic enzyme. Non-aqueous solvents also make it possible to run hydrolytic reactions in the reverse direction, forming a condensation product and water as a by-product. Non-aqueous solvents are being extensively used in cryoenzymology as antifreeze agents, in solubilizing and purifying enzymes, and to a lesser degree in two-phase systems in which the non-polar substrate is dissolved in the non-aqueous phase. Liquefied aqueous phenol is an extraordinary solvent for enzymes and other proteins. It is a powerful denaturant which rapidly and irreversibly extracts the enzyme into the phenol-rich phase of a phenol-water system. This property makes phenol useful for removing protein contaminants, and for detecting labile enzyme-substrate intermediates, by extracting substrate covalently bound to enzyme into the phenol-rich phase away from all other substrate, which generally remains in the aqueous phase.     

Enzyme activity in the crowded milieu

The cytosol of a cell is a concentrated milieu of a variety of different molecules, including small molecules (salts and metabolites) and macromolecules such as nucleic acids, polysaccharides, proteins and large macromolecular complexes. Macromolecular crowding in the cytosolic environment is proposed to influence various properties of proteins, including substrate binding affinity and enzymatic activity. Here we chose to use the synthetic crowding agent Ficoll, which is commonly used to mimic cytosolic crowding conditions to study the crowding effect on the catalytic properties of glycolytic enzymes, namely phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and acylphosphatase. We determined the kinetic parameters of these enzymes in the absence and in the presence of the crowding agent. We found that the Michaelis constant, K(m), and the catalytic turnover number, k(cat), of these enzymes are not perturbed by the presence of the crowding agent Ficoll. Our results support earlier findings which suggested that the Michaelis constant of certain enzymes evolved in consonance with the substrate concentration in the cell to allow effective enzyme function in bidirectional pathways. This conclusion is further supported by the analysis of nine other enzymes for which the K(m) values in the presence and absence of crowding agents have been measured.