The mechanism of the bond forming events in pyridine nucleotide linked oxidoreductases. Studies with epoxide inhibitors of lactic dehydrogenase and beta-hydroxybutyrate dehydrogenase.

2,3-Epoxybutyrate and 2,3-epoxypropionate act as effective competitive inhibitors of pig heart lactic dehydrogenase. KIapp for both inhibitors was pH dependent and varied according to the general equation KIapp = KI(1 +Ka/H+) which may be predicted if the binding of the epoxide to the E-NADH complex involves a compulsory protonation step. Values of KI(epoxybutyrate), KI(epoxypropionate) and pKa were estimated as 150 muM, 860 muM, and 6.8, respectively. The formation of an E-NADH epoxide inhibitor complex was followed directly by fluorescence measurements. Both epoxybutyrate and epoxypropionate enhanced fluorescence of the E-NADH complex and caused a 20-nm blue shift in the maximum emission wavelenght. The dissociation constants measured by fluorescence titration for both epoxides increased as the pH was raised reflecting a decreased affinity for the E-NADH complex. 2,3-Epoxybutyrate was also shown to inhibit beta-hydroxybutyrate dehydrogenase by a mechanism which is consistent with compulsory protonation prior to addition of the epoxide. These results are discussed in terms of a general mechanism for the bond forming events in pyridine nucleotide linked oxidore-ductases.     

Linear array of conserved sequence motifs to discriminate protein subfamilies: study on pyridine nucleotide-disulfide reductases

Background  

The pyridine nucleotide disulfide reductase (PNDR) is a large and heterogeneous protein family divided into two classes (I and II), which reflect the divergent evolution of its characteristic disulfide redox active site. However, not all the PNDR members fit into these categories and this suggests the need of further studies to achieve a more comprehensive classification of this complex family.     

The mitochondrial and prokaryotic proton-translocating NADH:ubiquinone oxidoreductases: similarities and dissimilarities of the quinone-junction sites

The catalytic properties of the rotenone-sensitive NADH:ubiquinone reductase (Complex I) in bovine heart submitochondrial particles and in inside-out vesicles derived from Paracoccus denitrificans and Rhodobacter capsulatus were compared. The prokaryotic enzymes catalyze the NADH oxidase and NADH:quinone reductase reactions with similar kinetic parameters as those for the mammalian Complex I, except for lower apparent affinities for the substrates--nucleotides. Unidirectional competitive inhibition of NADH oxidation by ADP-ribose, previously discovered for submitochondrial particles, was also evident for tightly coupled P. denitrificans vesicles, thus suggesting that a second, NAD(+)-specific site is present in the simpler prokaryotic enzyme. The inhibitor sensitivity of the forward and reverse electron transfer reactions was compared. In P. denitrificans and Bos taurus vesicles different sensitivities to rotenone and Triton X-100 for the forward and reverse electron transfer reactions were found. In bovine heart preparations, both reactions showed the same sensitivity to piericidin, and the inhibition was titrated as a straight line. In P. denitrificans, the forward and reverse reactions show different sensitivity to piericidin and the titrations of both activities were curvilinear with apparent I(50) (expressed as mole of inhibitor per mole of enzyme) independent of the enzyme concentration. This behavior is explained by a model involving two different sites rapidly interacting with piericidin within the hydrophobic phase.     

Expansion and evolution of insect GMC oxidoreductases

Background  

The GMC oxidoreductases comprise a large family of diverse FAD enzymes that share a homologous backbone. The relationship and origin of the GMC oxidoreductase genes, however, was unknown. Recent sequencing of entire genomes has allowed for the evolutionary analysis of the GMC oxidoreductase family.     

Dihydrolipoamide dehydrogenase from the halophilic archaebacterium Haloferax volcanii: characterization and N-terminal sequence.

Dihydrolipoamide dehydrogenase, a flavin disulfide reductase, has been purified and characterized from Haloferax volcanii. The enzyme is a dimer of relative mass 128,000, with an optimal activity at pH 9.0 in 1 M NaCl. Following reduction with its substrate, dihydrolipoamide, the enzyme is inactivated through covalent bond formation with the trivalent arsenical p-aminophenyl arsenoxide. The amino acid composition and the amino acid sequence of the first 49 residues of the N-terminus have been determined.     

Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12

Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher K(i) for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.     

The evolution of key functional floral traits in the early divergent angiosperm family Annonaceae

Potential key functional floral traits are assessed in the species‐rich early divergent angiosperm family Annonaceae. Pollinators (generally beetles) are attracted by various cues (particularly visual, olfactory, and thermogenic), with pollinators rewarded by nectar (generally as stigmatic exudate), heat, and protection within the partially enclosed floral chamber. Petals sometimes function as pollinator brood sites, although this could be deceptive. Annonaceae species are self‐compatible, with outcrossing promoted by a combination of protogyny, herkogamy, floral synchrony, and dicliny. Pollination efficiency is enhanced by pollen aggregation, changes in anthesis duration, and pollinator trapping involving a close alignment between petal movements and the circadian rhythms of pollinators. Most Annonaceae flowers are apocarpous, with syncarpy restricted to very few lineages; fertilization is therefore optimized by intercarpellary growth of pollen tubes, either by stigmatic exudate (suprastylar extragynoecial compitum) or possibly the floral receptacle (infrastylar extragynoecial compitum). Although Annonaceae lack a distinct style, the stigmas in several lineages are elongated to form “pseudostyles” that are hypothesized to function as sites for pollen competition. Flowers can be regarded as immature fruits in which the ovules are yet to be fertilized, with floral traits that may have little selective advantage during anthesis theoretically promoting fruit and seed dispersal. The plesiomorphic apocarpous trait may have been perpetuated in Annonaceae flowers as it promotes the independent dispersal of fruit monocarps (derived from separate carpels), thereby maximizing the spatial/temporal distance between seedlings. This might compensate for the lack of genetic diversity among seeds within fruits arising from the limited diversity of pollen donors.     

Dihydrolipoamide dehydrogenase from the halophilic archaeon Haloferax volcanii: homologous overexpression of the cloned gene.

The gene encoding dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been subcloned and overexpressed in the parent organism by using the halophilic archaeal rRNA promoter. The recombinant protein has been purified to homogeneity and characterized with respect to its kinetic, molecular, and salt-dependent properties. A dihydrolipoamide dehydrogenase-minus mutant of H. volcanii has been created by homologous recombination with the subcloned gene after insertion of the mevinolin resistance determinant into the protein-coding region. To explore the physiological function of the dihydrolipoamide dehydrogenase, the growth properties of the mutant halophile have been examined.     

Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia

The physiological function of the clostridial NADH- and NADPH-ferredoxin oxidoreductases was investigated with Clostridium pasteurianum and Clostridium butyricum.The NADH-ferredoxin oxidoreductases are concluded to be catabolic enzymes required for the reduction of ferredoxin by NADH. The conclusion is based on the finding that during the entire growth phase the fermentation of glucose can be formally represented by the weighted sum of Eqns 1 and 2, Glucose + 2 H₂O → 1 butyrate^? + 2 HCO₃^? + 3 H⁺ + 2 H₂ (1) Glucose + 4 H₂O → 2 acetate^? + 2 HCO₃^? + 4 H⁺ + 4 H₂ (2) and that in these redox processes NADH rather than NADPH is specifically formed during glyceraldehyde phosphate dehydrogenation. This NADH can be consumed by substrate reduction in Process 1 only, while it must be reoxidized in Process 2 by the ferredoxin-dependent proton reduction to hydrogen which involves the NADH-ferredoxin oxidoreductases.The kinetic and regulatory properties of these enzymes are in line with their catabolic role: they are found with high specific activities typical for other catabolic enzymes; essentially they catalyze electron flow from NADH to ferredoxin only because the back reaction is very effectively inhibited by low concentrations of NADH. These enzymes have a key role in the coupling of the two partial processes and in regulating the overall thermodynamic efficiency of the fermentations.The NADPH-ferredoxin oxidoreductases are concluded to participate in anabolism; they are required for the regeneration of NADPH. The conclusion is based on the finding that in the two clostridia all catabolic oxidations-reductions are specific for NAD(H) and that the usual NADPH-producing processes such as the glucose 6-phosphate dehydrogenase or malate enzyme reactions are absent. The kinetic properties of the enzymes are in agreement with their anabolic function: the NADPH-ferredoxin oxidoreductases are found with sufficient specific activities; they preferentially catalyze electron transfer from reduced ferredoxin to NADP⁺.     

The energetics and evolution of oxidoreductases in deep time

The core metabolic reactions of life drive electrons through a class of redox protein enzymes, the oxidoreductases. The energetics of electron flow is determined by the redox potentials of organic and inorganic cofactors as tuned by the protein environment. Understanding how protein structure affects oxidation–reduction energetics is crucial for studying metabolism, creating bioelectronic systems, and tracing the history of biological energy utilization on Earth. We constructed ProtReDox ( https://protein-redox-potential.web.app ), a manually curated database of experimentally determined redox potentials. With over 500 measurements, we can begin to identify how proteins modulate oxidation–reduction energetics across the tree of life. By mapping redox potentials onto networks of oxidoreductase fold evolution, we can infer the evolution of electron transfer energetics over deep time. ProtReDox is designed to include user-contributed submissions with the intention of making it a valuable resource for researchers in this field.     

Oxocarboxylic acids, pyridine nucleotide-linked oxidoreductases and serum factors in regulation of cell proliferation

When serum is made rate-limiting for clonal multiplication of human diploid fibroblasts, the presence of a 2-oxocarboxylic acid in the medium becomes essential. The requirement is independent of the 20 amino acids and glucose. Glyoxylic, pyruvic, 2-oxoglutaric, and oxalacetic acids are most effective. The types of 2-oxocarboxylic acids that support multiplication are oxidized substrates for several, pyridine nucleotide-linked intracellular oxidoreductases. The requirement is not satisfied by carboxylic acids, oxidized substrates for oxidoreductases that are not lniked to pyridine nucleotides, or by nonspecific electron acceptors. The quantitative requirement for 2-oxocarboxylic acids in cell multiplication is markedly affected by the concentration of serum proteins in the medium. Therefore, 2-oxocarboxylic acid metabolism may be related to the mechanism by which serum growth factors regulate cell multiplication.     

Ribonucleotide reductases: divergent evolution of an ancient enzyme

Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron–sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family.     

Convergent evolution and divergent selection: lizards at the White Sands ecotone

Ecological transition zones, where organismal phenotypes result from a delicate balance between selection and migration, highlight the interplay of local adaptation and gene flow. Here, I study the response of an entire species assemblage to natural selection across a common ecotone. Three lizard species, distributed along a dramatic environmental gradient in substrate color, display convergent adaptation of blanched coloration on the gypsum dunes of White Sands National Monument. I investigate the role of gene flow in modulating phenotypic response to selection by quantifying color variation and genetic variation across the ecotone. I find species differences in degree of background matching and in genetic connectivity of populations across the ecotone. Differences among species in phenotypic response to selection scale precisely to levels of genetic isolation. Species with higher levels of gene flow across the ecotone exhibit less dramatic responses to selection. Results also reveal a strong signal of ecologically mediated divergence for White Sands lizards. For all species, phenotypic variation is better explained by habitat similarity than genetic similarity. Convergent evolution of blanched coloration at White Sands clearly reflects the action of strong divergent selection; however, adaptive response appears to be modulated by gene flow and demographic history and can be predicted by divergence-with-gene-flow models.     

Pseudocyclical similarities and structural evolution of modular organisms

It was found that pseudocyclical similarities are common in modular organisms due to the peculiarities of their morphogenesis and ontogenesis and the system specifics of the modular organization. An analysis of the structural evolution in the different groups of modular living beings according to the concept of pseudocycles is topical, as it will contribute to the further development of evolutionary morphology and theoretical biology.