Functional evaluation of a nitrogenase-like protochlorophyllide reductase encoded by the chloroplast DNA of Physcomitrella patens in the cyanobacterium Leptolyngbya boryana

Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme consisting of the two components, L-protein (a ChlL dimer) and NB-protein (a ChlN-ChlB heterotetramer), to catalyze Pchlide reduction in Chl biosynthesis. While nitrogenase is distributed only among certain prokaryotes, the probable structural genes for DPOR are encoded by chloroplast DNA in lower plants. Here we show functional evaluation of DPOR encoded by chloroplast DNA in a moss Physcomitrella patens by the complementation analysis of the cyanobacterium Leptolyngbya boryana and the heterologous reconstitution of the moss L-protein and the cyanobacterial NB-protein. Two shuttle vectors to overexpress chlL and chlN-chlB from P. patens were introduced into the cyanobacterial chlL- and chlB-lacking mutants, respectively. Both transformants restored the ability to perform Chl biosynthesis in the dark, indicating that the chloroplast-encoded DPOR components form an active complex with the cyanobacterial components. The L-protein of P. patens was purified from the cyanobacterial transformant, and DPOR activity was reconstituted in a heterologous combination with the cyanobacterial NB-protein. The specific activity of the L-protein from P. patens was determined to be 118 nmol min(-1) mg (-1), which is even higher than that of the cyanobacterial L-protein (76 nmol min(-1) mg (-1)). Upon exposure to air, the activity of the L-protein from P. patens decayed with a half-life of 30 s, which was eight times faster than that of the cyanobacterial L-protein (240 s). These results suggested that the chloroplast-encoded L-protein functions as efficiently as the cyanobacterial L-protein but is more oxygen labile than the cyanobacterial L-protein.     

Isolation, characterization, and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria.

L-protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a lambda gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northern-blot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.     

Nitrogenase Fe protein-like Fe-S cluster is conserved in L-protein (BchL) of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus

Dark-operative protochlorophyllide reductase (DPOR) in bacteriochlorophyll biosynthesis is a nitrogenase-like enzyme consisting of L-protein (BchL-dimer) as a reductase component and NB-protein (BchN-BchB-heterotetramer) as a catalytic component. Metallocenters of DPOR have not been identified. Here we report that L-protein has an oxygen-sensitive [4Fe-4S] cluster similar to nitrogenase Fe protein. Purified L-protein from Rhodobacter capsulatus showed absorption spectra and an electron paramagnetic resonance signal indicative of a [4Fe-4S] cluster. The activity quickly disappeared upon exposure to air with a half-life of 20s. These results suggest that the electron transfer mechanism is conserved in nitrogenase Fe protein and DPOR L-protein.     

Functional expression of nitrogenase-like protochlorophyllide reductase from Rhodobacter capsulatus in Escherichia coli

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing D-ring reduction of protochlorophyllide in chlorophyll and bacteriochlorophyll biosynthesis. DPOR consists of two components, L-protein and NB-protein, which are structurally related to nitrogenase Fe-protein and MoFe-protein, respectively. Neither Fe-protein nor MoFe-protein is expressed as an active form in Escherichia coli due to the requirement of many Nif proteins for the assembly of the metallocenter and the maturation specific for diazotrophs. Here we report the functional expression of DPOR components from Rhodobacter capsulatus in Escherichia coli. Two overexpression plasmids for L-protein and NB-protein were constructed. L-protein and NB-protein purified from E. coli showed spectroscopic properties similar to those purified from R. capsulatus. L-protein and NB-protein activities were evaluated using a crude extract of E. coli overexpressing NB-protein and L-protein, respectively. Specific activities of the purified L-protein and NB-protein were 219+/-38 and 52.8+/-5.5 nmolChlorophyllide min(-1) mg(-1), respectively, which were even higher than those of L-protein and NB-protein purified from R. capsulatus. These E. coli strains provide a promising system for structural and kinetic analyses of the nitrogenase-like enzymes.     

Interaction between the lipoamide-containing H-protein and the lipoamide dehydrogenase (L-protein) of the glycine decarboxylase multienzyme system. 1. Biochemical studies.

Lipoamide dehydrogenase or dihydrolipoamide dehydrogenase (EC 1.8.1. 4) is the E3-protein component of the mitochondrial 2-oxoacid dehydrogenase multienzyme complexes. It is also the L-protein component of the glycine decarboxylase system. Although the enzymology of this enzyme has been studied exhaustively using free lipoamide as substrate, no data are available concerning the kinetic parameters of this enzyme with its physiological substrates, the dihydrolipoyl domain of the E2 component (dihydrolipoyl acyltransferase) of the 2-oxoacid dehydrogenase multienzyme complexes or the dihydrolipoyl H-protein of the mitochondrial glycine decarboxylase. In this paper, we demonstrate that Tris(2-carboxyethyl)phosphine, a specific disulfide reducing agent, allows a continuous reduction of the lipoyl group associated with the H-protein during the course of the reaction catalysed by the L-protein. This provided a valuable new tool with which to study the catalytic properties of the lipoamide dehydrogenase. The L-protein displayed a much higher affinity for the dihydrolipoyl H-protein than for free dihydrolipoamide. The oxidation of the dihydrolipoyl H-protein was not affected by the presence of structurally related analogues (apoH-protein or octanoylated H-protein). In marked contrast, these analogues strongly and competitively inhibited the decarboxylation of the glycine molecule catalysed by the P-protein component of the glycine decarboxylase system. Small unfolded proteolytic fragments of the H-protein, containing the lipoamide moiety, displayed Km values for the L-protein close to that found for the H-protein. On the other hand, these fragments were not able to promote the decarboxylation of the glycine in the presence of the P-protein. New highly hydrophilic lipoate analogues were synthesized. All of them showed Km and kcat/Km values very close to that found for the H-protein. From our results we concluded that no structural interaction is required for the L-protein to catalyse the oxidation of the dihydrolipoyl H-protein. We discuss the possibility that one function of the H-protein is to maintain a high concentration of the hydrophobic lipoate molecules in a nonmicellar state which would be accessible to the catalytic site of the lipoamide dehydrogenase.     

Identification of proteins in complexes with α-latrotoxin receptors

A thorough analysis of proteins capable of interacting with presynaptic receptors of α-latrotoxin was carried out. The protein components of receptor complexes were purified from rat brain membranes by the affinity chromatography on immobilized α-latrotoxin and antibodies to the cytoplasmic moiety of the calciumindependent receptor of α-latrotoxin (CIRL) and analyzed then by mass spectrometry. Several proteins were identified, with structural proteins, intracellular signal proteins, and proteins involved in the endocytosis and transport of synaptic vesicles being among them.     

Nicotinamide is a specific inhibitor of dark-operative protochlorophyllide oxidoreductase,a nitrogenase-like enzyme,from Rhodobacter capsulatus

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme consisting of two components, L-protein as a reductase component and NB-protein as a catalytic component. Elucidation of the crystal structures of NB-protein (Muraki et al., Nature 2010, 465: 110–114) has enabled us to study its reaction mechanism in combination with biochemical analysis. Here we demonstrate that nicotinamide (NA) inhibits DPOR activity by blocking the electron transfer from L-protein to NB-protein. A reaction scheme of DPOR, in which the binding of protochlorophyllide (Pchlide) to the NB-protein precedes the electron transfer from the L-protein, is proposed based on the NA effects.     

Scanning electron microscopy of amphibian lampbrush chromosomes

A protein having a molecular weight of 73,000 daltons has been purified from the nuclear membranes of preleptotene, leptotene, and zygotene cells. It has been named the leptotene protein (L-protein) because of its role in suppressing the replication of zygotene DNA sequences through leptotene until the initiation of zygotene DNA synthesis. The protein has been found to be highly specific in its inhibitory activity. S-phase replication in somatic nuclei and in microspore nuclei are unaffected by the protein. Only zygotene DNA sequences appear to be affected. L-protein binds specifically to zygotene DNA. The binding is limited to a relatively short DNA segment, probably no longer than 90 base pairs (bp). Chloroplast and mitochondrial DNA do not bind to the protein, but a low level of binding is displayed by DNA from several other eukaryotic species. The L-protein also has the capacity to nick the bound DNA in the presence of ATP. Nicking does not occur in the absence of binding. Using supercoiled plasmids with zygotene DNA inserts as substrates, the nicking has been found to be confined to a small region of the plasmid and to occur in only one of the strands. The L-protein is considered to be one of the principal factors responsible for the irreversible commitment of cells to meiosis at the end of the preleptotene S-phase. It is also proposed that its endonucleolytic activity plays a role in the initiation of synapsis.     

Interaction between the lipoamide-containing H-protein and the lipoamide dehydrogenase (L-protein) of the glycine decarboxylase multienzyme system 2. Crystal structures of H- and L-proteins.

The glycine decarboxylase complex consists of four different component enzymes (P-, H-, T- and L-proteins). The 14-kDa lipoamide-containing H-protein plays a pivotal role in the complete sequence of reactions as its prosthetic group (lipoic acid) interacts successively with the three other components of the complex and undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. With the aim to understand the interaction between the H-protein and its different partners, we have previously determined the crystal structure of the oxidized and methylaminated forms of the H-protein. In the present study, we have crystallized the H-protein in its reduced state and the L-protein (lipoamide dehydrogenase or dihydrolipoamide dehydrogenase). The L-protein has been overexpressed in Escherichia coli and refolded from inclusion bodies in an active form. Crystals were obtained from the refolded L-protein and the structure has been determined by X-ray crystallography. This first crystal structure of a plant dihydrolipoamide dehydrogenase is similar to other known dihydrolipoamide dehydrogenase structures. The crystal structure of the H-protein in its reduced form has been determined and compared to the structure of the other forms of the protein. It is isomorphous to the structure of the oxidized form. In contrast with methylaminated H-protein where the loaded lipoamide arm was locked into a cavity of the protein, the reduced lipoamide arm appeared freely exposed to the solvent. Such a freedom is required to allow its targeting inside the hollow active site of L-protein. Our results strongly suggest that a direct interaction between the H- and L-proteins is not necessary for the reoxidation of the reduced lipoamide arm bound to the H-protein. This hypothesis is supported by biochemical data [Neuburger, M., Polidori, A.M., Piètre, E., Faure, M., Jourdain, A., Bourguignon, J., Pucci, B. & Douce, R. (2000) Eur. J. Biochem. 267, 2882-2889] and by small angle X-ray scattering experiments reported herein.     

β-Bungarotoxin antagonizes the effect of α-latrotoxin from black widow spider venom on the neuromuscular junction

Sustained contraction of the chick biventer cervicis nerve-muscle preparations evoked by α-latrotoxin was antagonized quickly by β-bungarotoxin. This effect of β-bungarotoxin was dependent on its phospholipase A₂ activity. In contrast, pancreatic phospholipase A₂ was ineffective even at a much higher dose. It is concluded that α-latrotoxin needs intact presynaptic membrane to exert its effect.     

NB-protein (BchN-BchB) of dark-operative protochlorophyllide reductase is the catalytic component containing oxygen-tolerant Fe-S clusters

Dark-operative protochlorophyllide (Pchlide) oxidoreductase is a nitrogenase-like enzyme consisting of the two components, L-protein (BchL-dimer) and NB-protein (BchN-BchB-heterotetramer). Here, we show that NB-protein is the catalytic component with Fe-S clusters. NB-protein purified from Rhodobacter capsulatus bound Pchlide that was readily converted to chlorophyllide a upon the addition of L-protein and Mg-ATP. The activity of NB-protein was resistant to the exposure to air. A Pchlide-free form of NB-protein purified from a bchH-lacking mutant showed an absorption spectrum suggesting the presence of Fe-S centers. Together with the Fe and sulfide contents, these findings suggested that NB-protein carries two oxygen-tolerant [4Fe-4S] clusters.     

Overexpression and characterization of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus

Dark-operative protochlorophyllide oxidoreductase (DPOR) plays a crucial role in light-independent (bacterio)chlorophyll biosynthesis in most photosynthetic organisms. However, the biochemical properties of DPOR are still largely undefined. Here, we constructed an overexpression system of two separable components of DPOR, L-protein (BchL) and NB-protein (BchN-BchB), in the broad-host-range vector pJRD215 in Rhodobacter capsulatus. We established a stable DPOR assay system by mixing crude extracts from the two transconjugants under anaerobic conditions. Using this assay system, we demonstrated some basic properties of DPOR. The Km value for protochlorophyllide was 10.6 μM. Ferredoxin functioned as an electron donor to DPOR. Elution profiles in gel filtration chromatography indicated that L-protein and NB-protein are a homodimer [(BchL)₂] and a heterotetramer [(BchN)₂(BchB)₂], respectively. These results provide a framework for the characterization of these components in detail, and further support a nitrogenase model of DPOR.     

Effects of α-latrotoxin on synaptic transmission between identified neurons in the snail central nervous system

The effects of -latrotoxin — a component of black widow spider venom — on an identified monosynaptic peptidergic synapse were investigated in the central nervous system ofHelix pomatia. Extracellular and intracellular application of the toxin was found to increase and block postsynaptic current respectively. Current induced by intracellular injection of cAMP which imitates postsynaptic current was inhibited, irrespective of the mode of toxin application. It was concluded on the basis of the experiments performed that -latrotoxin manifests its reinforcing effect, acting on the transmitter release system in the presynaptic neuron. It can also induce an inhibitory effect, however, operating on the postsynaptic cell direct — an effect which may be mediated by the cAMP system.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. A. A. Bogomolets Medical Institute of Biochemistry, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 430–437, July–August, 1992.     

Presynaptic Inhibition by Concanavalin A: Are α-Latrotoxin Receptors Involved in Action Potential-Dependent Transmitter Release?

Abstract: Effects of concanavalin A on transmitter release were investigated in primary cultures of chick sympathetic neurons. The lectin reduced electrically evoked [³H]noradrenaline release by up to 30% with half-maximal inhibition at 0.16 µ M . Concanavalin A also reduced the release triggered by extracellular Ca²⁺ in neurons depolarized by 25 m M K⁺ or rendered Ca²⁺-permeable by the ionophore A23187. The inhibitory action of concanavalin A on electrically evoked release was additive to that of the α₂-adrenergic agonist UK 14,304. Inactivation of G_s and G_i/G_o type G proteins by either cholera or pertussis toxin did not alter the inhibitory effect of the lectin. Concanavalin A failed to affect the resting membrane potential, action potential waveforms, or voltage-dependent K⁺ and Ca²⁺ currents. In contrast, the lectin efficiently blocked both the Ca²⁺-dependent and -independent α-latrotoxin-induced transmitter release, but only when applied before the toxin. The reduction of electrically evoked, as well as α-latrotoxin-evoked, release by concanavalin A was attenuated in the presence of glucose and abolished by methyl α- d -mannopyranoside. The dimeric derivative, succinyl-concanavalin A, was significantly less active than tetrameric concanavalin A. In bovine adrenal chromaffin cells, which displayed only weak secretory responses to α-latrotoxin, concanavalin A failed to alter K⁺-evoked catecholamine secretion. These results show that concanavalin A causes presynaptic inhibition in sympathetic neurons and indicate that cross-linking of α-latrotoxin receptors may reduce action potential-dependent transmitter release.     

Dual topology of the large envelope protein of duck hepatitis B virus: determinants preventing pre-S translocation and glycosylation.

The biosynthesis and topology of the large envelope protein (L protein) of hepadnaviruses was investigated using the duck hepatitis B virus (DHBV) model, which also allows the study of hepadnavirus morphogenesis in experimentally infected hepatocytes. Results from proteolysis of virus particles and from the analysis of topology and posttranslational modification of L chains synthesized in vivo or in a cell-free system both support the presence of a mixed population of L-protein molecules with their N-terminal pre-S domain located either inside or outside the virus particle. During L biosynthesis and DHBV morphogenesis, pre-S, together with the neighboring transmembrane domain (TM-I), initially remained cytoplasmically disposed and was translocated only posttranslationally. Delayed pre-S translocation into a post-endoplasmic reticulum compartment is also indicated by the absence of glycosylation at a modification-competent pre-S glycosylation site. Major features of L-protein biosynthesis and of the resulting dual topology appear to be conserved between avian and mammalian hepadnaviruses, supporting the model that pre-S domains function in part either as an internal matrix for capsid envelopment or externally as a ligand for cellular receptor binding. However, differences in the mechanisms controlling pre-S translocation were revealed by the results of mutational analyses identifying and characterizing cis-acting determinants in pre-S that delay its cotranslational translocation. Our data from DHBV demonstrate the negative influence of a cluster of positively charged amino acid residues next to TM-I, a motif that is conserved among the avian but absent from mammalian hepadnaviruses. Additional control elements, which are apparently shared between both virus groups and which may serve in chaperone binding, were mapped by deletion analysis in the central part of pre-S.     

Effect of Latrotoxin-Like Protein on Spontaneous Postsynaptic Activity in Hippocampal Cell Cultures

-Latrotoxin, an active component of black widow spider venom, is known to enhance spontaneous neurotransmitter release. In cultured rat hippocampal neurons, we studied the effects of latrotoxin-like protein (protein purified from the bovine brain and exhibiting some functional properties similar to those of -latrotoxin) on spontaneous GABA-ergic inhibitory currents (IPSC). Latrotoxin-like protein was found to dramatically increase the frequency of spontaneous IPSC recorded in cell cultures of dissociated hippocampal neurons in the presence of tetrodotoxin. Possible mechanisms of the action of latrotoxin-like protein on transmitter release are discussed.     

α-Latrotoxin: preparation and effects on calcium fluxes

Abstract A toxin that causes a massive presynaptic activation of transmitter release from nerve terminals is α-latrotoxin, isolated from Latrodectus tredecimguttatus spider venom. This toxin has been highly purified, utilizing as a biological assay a toxin-dependent increase in ⁴⁵Ca²⁺-accumulation by PC12 cells. The purification protocol includes an ion-exchange step and a gel-filtration column, by fast-flow liquid chromatography. The resulting toxin is a polypeptide of about 125 kDa in molecular mass. At nmol concentrations it specifically activates calcium influx and transmitter secretion after interacting with neuronal acceptors of the presynaptic membrane. The inhibitory effect of trivalent ions (which may develop as degradation product of ⁴⁵Ca²⁺) on toxin-dependent calcium accumulation by PC12 cells is described. The results obtained suggest that calcium fluxes directly involved in the neurosecretory event, may occur through newly formed toxin-dependent channels.     

Effect of thiamine on the processes responsible for acetylcholine secretion in the frog neuromuscular synapses

The effect of vitamin B₁ (thiamine, 10^–10–10^–3 M) on direct (transmitter secretion) and recurrent (resynthesis of the transmitter and its storage in synaptic vesicles) processes of acetylcholine (ACh) secretion was studied in the frog neuromuscular junction. In Ca²⁺-containing extracellular medium, the facilitatory effects of thiamine and -latrotoxin (an increase in the frequency of miniature end-plate potentials, MEPP) were additive, regardless of the sequence of their application. After partial exhaustion of the synaptic vesicle stores caused by -latrotoxin (2 nM) in Ca²⁺-free extracellular medium, thiamine accelerated the Ca²⁺-induced recovery of the ACh secretion. In the presence of thiamine, there were two phases in the dependence of quantum content of an end-plate potential (EPP) on stimulation frequency, which are typical of the effects of Sr²⁺ and Ba²⁺ on the ACh secretion. Under conditions of depression and postdepression recovery, the effect of thiamine on the time course of the changes in EPP amplitude was similar to that produced by Ba²⁺. Possible mechanisms of the effects of vitamin B₁ on the processes responsible for the ACh secretion and the dependence of the MEPP frequency on the concentrations of thiamine and thiamine diphosphate are discussed in light of the above results.Neirofiziologiya/Neurophysiology, Vol. 26, No. 4, pp. 291–298, July–August, 1994.