Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction

Nitrogen is a vital component in living organisms as it participates in the making of essential biomolecules such as proteins, nucleic acids, etc. In the biosphere, nitrogen cycles between the oxidation states +V and -III producing many species that constitute the biogeochemical cycle of nitrogen. All reductive branches of this cycle involve the conversion of nitrate to nitrite, which is catalyzed by the enzyme nitrate reductase. The characterization of nitrate reductases from prokaryotic organisms has allowed us to gain considerable information on the molecular basis of nitrate reduction. Prokaryotic nitrate reductases are mononuclear Mo-containing enzymes sub-grouped as respiratory nitrate reductases, periplasmic nitrate reductases and assimilatory nitrate reductases. We review here the biological and molecular properties of these three enzymes along with their gene organization and expression, which are necessary to understand the biological processes involved in nitrate reduction.     

Look on the positive side! The orientation, identification and bioenergetics of 'Archaeal' membrane-bound nitrate reductases

Many species of Bacteria and Archaea respire nitrate using a molybdenum-dependent membrane-bound respiratory system called Nar. Classically, the 'Bacterial' Nar system is oriented such that nitrate reduction takes place on the inside of this membrane. However, the active site subunit of the 'Archaeal' Nar systems has a twin arginine ('RR') motif, which is a suggestion of translocation to the outside of the cytoplasmic membrane. These 'Archaeal' type of nitrate reductases are part of a group of molybdoenzymes with an 'RR' motif that are predicted to have an aspartate ligand to the molybdenum ion. This group includes selenate reductases and possible sequence signatures are described that serve to distinguish the Nar nitrate reductases from the selenate reductases. The 'RR' sequences of nitrate reductases of Archaea and some that have recently emerged in Bacteria are also considered and it is concluded that there is good evidence for there being both Archaeal and Bacterial examples of Nar-type nitrate reductases with an active site on the outside of the cytoplasmic membrane. Finally, the bioenergetic consequences of nitrate reduction on the outside of the cytoplasmic membrane have been explored.     

Ketopantoic acid and ketopantoyl lactone reductases. Stereospecificity of transfer of hydrogen from reduced nicotinamide adenine dinucleotide phosphate.

The stereospecificity of hydrogen transfer from NADPH to the appropriate carbonyl substrate catalyzed by ketopantoic acid and ketopantoyl acid and ketopantoyl lactone reductases of yeast (Saccharomyces cerevisiae) and Escherichia coli has been determined. Yeast and E. coli ketopantoic acid reductases are B-specific enzymes which transfer hydrogen from [4B-3H]-NADPH to ketopantoic acid to form [3H]pantoic acid. In contrast to the usual observations on the stereospecificity of functionally similar dehydrogenases from different species, yeast and E. coli ketopantoyl lactone reductases exhibit opposite stereospecificities. Both of two forms of yeast ketopantoyl lactone reductases are A-specific enzymes which form [3H]pantoyl lactone from ketopantoyl lactone and [4A-3H]NADPH, whereas, two forms of E. coli ketopantoyl lactone reductases are B-specific enzymes.     

Octaheme nitrite reductases: Structure and properties

Octaheme oxidoreductases are widespread among various bacterial taxa involved in the biogeochemical nitrogen cycle. The evolution of octaheme oxidoreductases of the nitrogen cycle from the evolutionarily more ancient pentaheme nitrite reductases was accompanied by changes in function from reduction of nitrogen oxides to their oxidation under changing environmental conditions. Octaheme nitrite reductases, which are the subject of the present review, are of a transitional form that combines structural and functional characteristics of pentaheme reductases and octaheme oxidases and possesses a number of unique features typical of only this family of enzymes. The review summarizes data on structure-function investigations of the family of octaheme nitrite reductases. Emphasis is given to comparison of the structures and functions of octaheme nitrite reductases and other multiheme oxidoreductases of the nitrogen cycle.     

Localization of the cytochrome cd1 and copper nitrite reductases in denitrifying bacteria.

The locations of cytochrome cd1 nitrite reductases in Pseudomonas aeruginosa and Pseudomonas fluorescens and copper nitrite reductases in Achromobacter cycloclastes and Achromobacter xylosoxidans were identified. Immunogold labeling with colloidal-gold probes showed that the nitrite reductases were synthesized exclusively in anaerobically grown (denitrifying) cells. Little immunogold label occurred in the cytoplasm of these four strains; most was found in the periplasmic space or was associated with cell membranes. Immunogold labeling of thin sections was superior to fractionation by osmotic shock for locating nitrite reductases. The results support models of dentrification energetics that require a periplasmic, not a cytoplasmic, location for nitrite reductases.     

Dihydrodipicolinate reductases from Bacillus cereus and Bacillus megaterium.

Dyhydrodipicolinate reductases were purified 100-fold from crude extracts of B. cereus and B. megaterium and their properties were compared with those of the reductase from B. subtilis. The molecular weights of the reductases of B. cereus and B. megaterium were fount to be 155,000 and 150,000, respectively. These reductases were shown to be free of flavin, unlike the B. subtilis enzyme, which contains flavin. Both NADPH and NADH acted as coenzymes for these two reductases. NADPH being three or four times more effective than NADH. The Km values for NADPH and dihydrodipicolinate were 8 micrometer and 62 micrometer, respectively, with B. cereus reductase, and 13 micrometer and 59 micrometer with B. megaterium reductase. The pH optima of the enzymes from B. cereus and B. megaterium were pH 7.4 and 7.2, respectively. The reductases were inhibited by dipicolinate noncompetitively with respect to dihydrodipicolinate and the Ki values were 85 micrometer and 140 micrometer, respectively. Lysine and diaminopimelate were not inhibitory. The properties of the reductases from B. cereus and B. megaterium were similar, but they differed considerably from those of the B. subtilis enzyme. However, all three Bacillus reductases were markedly inhibited by dipicolinate, unlike the enzyme from E. coli.     

Functional domains of assimilatory nitrate reductases and nitrite reductases

Biochemical investigation of nitrate assimilation enzymes spans the past four decades. With the molecular cloning of genes for nitrate reductases and nitrite reductases, exciting new prospects are developing for the study of these enzymes. As large, complex enzymes with multiple redox centers, these two types of reductases should help us gain understanding of structural, functional and evolutionary relationships among the diverse group of multicenter redox enzymes.     

Nitrate reductases: Structure,functions, and effect of stress factors

Structural and functional peculiarities of four types of nitrate reductases are considered: assimilatory nitrate reductase of eukaryotes, as well as cytoplasmic assimilatory, membrane-bound respiratory, and periplasmic dissimilatory bacterial nitrate reductases. Arguments are presented showing that eukaryotic organisms are capable of nitrate dissimilation. Data concerning new classes of extremophil nitrate reductases, whose active center does not contain molybdocofactor, are summarized.     

Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases

Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.     

Microbial ferric iron reductases

Almost all organisms require iron for enzymes involved in essential cellular reactions. Aerobic microbes living at neutral or alkaline pH encounter poor iron availability due to the insolubility of ferric iron. Assimilatory ferric reductases are essential components of the iron assimilatory pathway that generate the more soluble ferrous iron, which is then incorporated into cellular proteins. Dissimilatory ferric reductases are essential terminal reductases of the iron respiratory pathway in iron-reducing bacteria. While our understanding of dissimilatory ferric reductases is still limited, it is clear that these enzymes are distinct from the assimilatory-type ferric reductases. Research over the last 10 years has revealed that most bacterial assimilatory ferric reductases are flavin reductases, which can serve several physiological roles. This article reviews the physiological function and structure of assimilatory and dissimilatory ferric reductases present in the Bacteria, Archaea and Yeast. Ferric reductases do not form a single family, but appear to be distinct enzymes suggesting that several independent strategies for iron reduction may have evolved.     

The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases.

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."     

Comparison of the properties of detergent-solubilized NADPH-cytochrome P-450 reductases from pig liver and kidney immunochemical,kinetic, and reconstitutive properties

NADPH-cytochrome P-450 reductases from pig liver and kidney and rabbit liver microsomes were purified to a specific activity of 50–62 μmol cytochrome c reduced/min/mg. All reductase preparations were separated into one major and one minor fraction on Sephadex G-200 columns. The molecular weights of the major fractions of the reductases were estimated to be 74,000, 75,000, and 75,500 for rabbit liver, pig kidney, and liver reductases, respectively, whereas the molecular weight of the minor fractions of these reductases, 67,000, was the same as that of the steapsin-solubilized pig liver reductase on SDS-polyacrylamide gel electrophoresis. K_m values for NADPH and cytochrome c were: 20 and 29 μm or 14 and 28 μm for the pig kidney or liver reductase, respectively. Immunochemical studies, including Ouchterlony double diffusion experiments and inhibition of benzphetamine N-demethylation activity in microsomes by antibody against pig liver NADPH-cytochrome P-450 reductase, indicated the similarity of the purified liver and kidney reductases. There were no differences in the ability to reconstitute NADPH-mediated benzphetamine N-demethylation and laurate hydroxylation in reconstituted systems between the pig liver and kidney reductases, indicating that the reductase did not determine substrate specificity in these systems.     

Isolation and characterization of indole-3-acetaldehyde reductases from Cucumis sativus.

In a continuing study of the biosynthetic pathway and regulatory mechanisms governing indole-3-acetic acid (auxin) formation, we report the isolation and initial characterization of three distinct indole-3-acetaldehyde reductases from cucumber seedlings. These enzymes catalyze the reduction of indole-3-acetaldehyde to indole-3-ethanol with the concomitant oxidation of NAD(P)H to NAD(P)+. Two of the reductases are specific for NADPH as second substrate, while the third is specific for NADH. The enzymes show a strong specificity for indoleacetaldehyde, with apparent Km values of 73mum, 130mum, and 400mum being calculated for the two NADPH-specific reductases and the NADH-specific reductase, respectively. Under no conditions of substrate concentration, incubation time, or assay method could the reverse reaction be observed. Chromatography on a calibrated Sephadex gel column led to estimated molecualr weights of 52,000 and 17,000 for the NADPH-specific reductases, while a value of 33,000 was obtained for the NADH-specific reductase. Both NADPH-specific reductases showed a pH optimum of 5.2 with a secondary optimum at 7.0, and both enzymes were activated by increasing ionic strength. The NADH-specific reductase showed a pH optimum of 7.0 with a secondary optimum at 6.1 and was slightly inhibited by increasing ionic strength.     

Comparative analysis of cytosolic and solubilized mitochondrial menadione reductases from rat liver]

The kinetic properties of cytosolic and solubilized mitochondrial menadione reductases (EC 1.6.99.2) from rat liver were compared. The mechanism of the reaction of cytosolic and mitochondrial menadione reductases with NADH and 4-anilino-5-methoxy-1,2-benzoquinone (AMOBQ) as substrates obeys the "ping-pong" kinetics. AMOBQ is a competitive inhibitor of cytosolic menadione reductase (Ki = 219 microM). Both menadione reductases have similar or identical values of true and effective kinetic constants and similar electrophoretic mobilities.     

Immunological identification of soluble carbonyl reductase with testosterone 17β-dehydrogenase (NADP+) in guinea pig liver and kidney

Antiserum was produced against one of two carbonyl reductases purified from guinea pig liver cytosol to identify the enzymes as testosterone 17β-dehydrogenase isozymes. Immunoelectrophoresis and immunoprecipitation with the antiserum indicated that the two reductases had common antigenic sites. The antiserum inhibited most of both carbonyl reductase and testosterone 17β-dehydrogenase activities in the purified reductases and in cytosols of liver and kidney.     

Electron donation between copper containing nitrite reductases and cupredoxins: the nature of protein-protein interaction in complex formation.

In denitrifying organisms with copper containing dissimilatory nitrite reductases, electron donation from a reduced cupredoxin is an essential step in the reduction of nitrite to nitric oxide. Copper nitrite reductases are categorised into two subgroups based on their colour, green and blue, which are found in organisms where the cupredoxins are pseudoazurins and azurins, respectively. In view of this and some in vitro electron donation experiments, it has been suggested that copper nitrite reductases have specific electron donors and that electron transfer takes place in a specific complex of the two proteins. We report results from the first comprehensive electron donation experiments using three copper nitrite reductases, one green and two blue, and five cupredoxins, one pseudoazurin and four azurins. Our data show that pseudoazurin can readily donate electrons to both blue and green copper nitrite reductases. In contrast, all of the azurins react very sluggishly as electron donors to the green nitrite reductase. These results are discussed in terms of surface compatibility of the component proteins, complex formation, overall charges, charge distribution, hydrophobic patches and redox potentials. A docking model for the complexes is proposed.     

Compositional relatedness of aldehyde reductases from several species

Summary The amino acid compositions of several monomeric NADPH-dependent aldehyde reductases from a variety of species have been determined and analyzed by the difference index method of Metzger et al. (1968). The difference indexes among mammals range from 4.15 – 6.10 indicating considerable homology. Comparison of chicken aldehyde reductase with mammalian aldehyde reductases gave values in the range 6.8 – 9.9 suggesting a close relationship whereas the difference indexes for the enzymes from fruit fly and Baker's yeast versus vertebrate aldehyde reductases (10.9 – 14.4) indicate more distant relationships. The extent of sequence homology among aldehyde reductases from these species was estimated from a plot of difference index versus percent sequence difference for oxido-reductases of known sequence. From this plot, and using a mammal-chicken divergence time of 300 million years and a mammalian order split of 75 million years, the rate of evolution of aldehyde reductases was calculated to lie in the range 5.8 – 15.6% sequence difference per 100 million years. Comparison with rates of evolution of oligomeric dehydrogenases indicates that aldehyde reductases comprise the most rapidly evolving family of oxido-reductases. This is probably related to the monomericity of aldehyde reductases since there is a direct correlation between the number of subunits and the rate of evolution.     

Imine Reductases: A Comparison of Glutamate Dehydrogenase to Ketimine Reductases in the Brain

A key intermediate in the glutamate dehydrogenase (GDH)-catalyzed reaction is an imine. Mechanistically, therefore, GDH exhibits similarities to the ketimine reductases. In the current review, we briefly discuss (a) the metabolic importance of the GDH reaction in liver and brain, (b) the mechanistic similarities between GDH and the ketimine reductases, (c) the metabolic importance of the brain ketimine reductases, and (d) the neurochemical consequences of defective ketimine reductases. Our review contains many historical references to the early work on amino acid metabolism. This work tends to be overlooked nowadays, but is crucial for a contemporary understanding of the central importance of ketimines in nitrogen and intermediary metabolism. The ketimine reductases are important enzymes linking nitrogen flow among several key amino acids, yet have been little studied. The cerebral importance of the ketimine reductases is an area of biomedical research that deserves far more attention.     

Structural and mechanistic insights on nitrate reductases

Nitrate reductases (NR) belong to the DMSO reductase family of Mo‐containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane‐bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data.     

Proton pathways, ligand binding and dynamics of the catalytic site in haem-copper oxygen reductases: a comparison between the three families

Haem-copper oxygen reductases are the widest spread enzymes involved in aerobic respiratory chains, in Eukarya, Bacteria and Archaea. However, both the catalytic mechanism for oxygen reduction and its coupling to proton translocation remain to be fully understood. In this article we analyse the experimental data gathered in recent years for haem-copper reductases presenting features distinct from the mitochondrial-like enzymes. These features further support the classification of several families of haem-copper oxygen reductases based on their proton pathways and previously proposed by us [Biochim. Biophys. Acta 1505 (2001) 185], and allow to identify the minimal essential elements for these enzymes.