Dimeric Holoenzyme Research Articles

Protein Phosphatase 2A Is Regulated by Protein Kinase Cα (PKCα)-dependent Phosphorylation of Its Targeting Subunit B56α at Ser41

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser(41) of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca(2+) release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser(41). This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca(2+) homeostasis.

Subunit composition of CP43-less photosystem II complexes ofSynechocystissp. PCC 6803: implications for the assembly and repair of photosystem II

Photosystem II (PSII) mutants are useful experimental tools to trap potential intermediates involved in the assembly of the oxygen-evolving PSII complex. Here, we focus on the subunit composition of the RC47 assembly complex that accumulates in a psbC null mutant of the cyanobacterium Synechocystis sp. PCC 6803 unable to make the CP43 apopolypeptide. By using native gel electrophoresis, we showed that RC47 is heterogeneous and mainly found as a monomer of 220 kDa. RC47 complexes co-purify with small Cab-like proteins (ScpC and/or ScpD) and with Psb28 and its homologue Psb28-2. Analysis of isolated His-tagged RC47 indicated the presence of D1, D2, the CP47 apopolypeptide, plus nine of the 13 low-molecular-mass (LMM) subunits found in the PSII holoenzyme, including PsbL, PsbM and PsbT, which lie at the interface between the two momomers in the dimeric holoenzyme. Not detected were the LMM subunits (PsbK, PsbZ, Psb30 and PsbJ) located in the vicinity of CP43 in the holoenzyme. The photochemical activity of isolated RC47-His complexes, including the rate of reduction of P680+, was similar to that of PSII complexes lacking the Mn4CaO5 cluster. The implications of our results for the assembly and repair of PSII in vivo are discussed.

Expression in Escherichia coli of the catalytic domain of human proline oxidase

The human PRODH gene has been shown to have unique roles in regulating cell survival and apoptotic pathways and it has been related to velocardiofacial syndrome/DiGeorge syndrome and increased susceptibility to schizophrenia. It encodes for the flavoprotein proline oxidase (PO), which catalyzes the conversion of l-proline to Δ1-pyrroline-5-carboxylate. Despite the important physiological and medical interest in human PO, up to now only microbial homologues of PO have been expressed as recombinant protein and fully characterized. By using a bioinformatics analysis aimed at identifying the catalytic domain and the regions with a high intrinsic propensity to structural disorder, we designed deletion variants of human PO that were successfully expressed in Escherichia coli as soluble proteins in fairly high amounts (up to 10mg/L of fermentation broth). The His-tagged PO-barrelN protein was isolated as an active (the specific activity is 0.032U/mg protein), dimeric holoenzyme showing the typical spectral properties of FAD-containing flavoprotein oxidases. These results pave the way for elucidating structure–function relationships of this human flavoenzyme and clarifying the effect of the reported polymorphisms associated with disease states.

Rapid Induction of Cell Death by Selenium-compromised Thioredoxin Reductase 1 but Not by the Fully Active Enzyme Containing Selenocysteine

Mammalian thioredoxin reductases are selenoproteins. For native catalytic activity, these enzymes utilize a C-terminal -Gly-Cys-Sec-Gly-COOH sequence (where Sec is selenocysteine) forming a redox active selenenylsulfide/selenolthiol motif. A range of cellular systems depend upon or are regulated by thioredoxin reductase and its major protein substrate thioredoxin, including apoptosis signal-regulating kinase 1, peroxiredoxins, methionine sulfoxide reductase, and several transcription factors. Cytosolic thioredoxin reductase 1 (TrxR1) is moreover inhibited by various electrophilic anticancer compounds. TrxR1 is hence generally considered to promote cell viability. However, several recent studies have suggested that TrxR1 may promote apoptosis, and the enzyme was identified as GRIM-12 (gene associated with retinoid interferon-induced mortality 12). Transient transfection with GRIM-12/TrxR1 was also shown to directly induce cell death. To further analyze such effects, we have here employed lipid-mediated delivery of recombinant TrxR1 preparations into human A549 cells, thereby bypassing selenoprotein translation to facilitate assessment of the protein-related effects on cell viability. We found that selenium-deficient TrxR1, having a two-amino acid-truncated C-terminal -Gly-Cys-COOH motif, rapidly induced cell death (38 +/- 29% apoptotic cells after 4 h; p < 0.005 compared with controls). Cell death induction was also promoted by selenium-compromised TrxR1 derivatized with either cis-diamminedichloroplatinum (II) (cisplatin) or dinitrophenyl moieties but not by the structurally related non-selenoprotein glutathione reductase. In contrast, TrxR1 with intact selenocysteine could not promote cell death. The direct cellular effects of selenium-compromised forms of TrxR1 may be important for the pathophysiology of selenium deficiency as well as for the efficacy of antiproliferative drugs targeting the selenocysteine moiety of this enzyme.

Dimeric and monomeric Bacillus subtilis RNase P holoenzyme in the absence and presence of pre-tRNA substrates.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that catalyzes the 5' maturation of tRNA precursors. The bacterial RNase P holoenzyme is composed of a large, catalytic RNA and a small protein. Our previous work showed that Bacillus subtilis RNase P forms a specific "dimer" that contains two RNase P RNA and two RNase P protein subunits in the absence of substrate. We investigated the equilibrium and the structure of the dimeric and the monomeric holoenzyme in the absence and presence of substrates by synchrotron small-angle X-ray scattering, 3' autolytic processing, and hydroxyl radical protection. In the absence of substrate, the dimer-monomer equilibrium is sensitive to monovalent ions and the total holoenzyme concentration. At 0.1 M NH4Cl, formation of the dimer is strongly favored, whereas at 0.8 M NH4Cl, the holoenzyme is a monomer. Primary hydroxyl radical protection in the dimer is located in the specificity domain, or domain I, of the RNase P RNA. The ES complex with a substrate containing a single tRNA is always monomeric. In contrast, the dominant ES complex with a substrate containing two tRNAs is dimeric at 0.1 M NH4Cl and monomeric at 0.8 M NH4Cl. Our results show that the B. subtilis holoenzyme can be a dimer and a monomer, and the fraction of the dimer is very sensitive to the environment. Under a variety of conditions, both the holoenzyme dimer and monomer can be present in significant amounts. Because the majority of tRNA genes are organized in large operons and because of the lack of RNase E in B. subtilis, a dimeric holoenzyme may be necessary to facilitate the processing of large precursor tRNA transcripts. Alternatively, the presence of two forms of the RNase P holoenzyme may be required for other yet unknown functions.

UDP-Galactose 4-Epimerase from Escherichia coli: Formation of Catalytic Site during Reversible Folding

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of molecular weight 39 kDa/subunit having noncovalently bound NAD acting as cofactor. Denaturation by 8 M urea at pH 7.0 causes 85% loss of its secondary structure and dissociation of its constituent molecules. Dilution of the denaturant by buffer at pH 8.5 leads to functional reconstitution of the dimeric holoenzyme. The refolding process is biphasic: after 2 min an equilibrium conformer is formed having 72% of its native secondary structure and by 60 min reactivation becomes complete. The early intermediate has lower energy of activation against thermal denaturation than the reactivated state. Patterns of trypsin digestion suggests a native like structure of this intermediate. Variation of solvent viscosity and ionic strength and inclusion of proline cis-trans isomerase in the refolding process do not alter kinetics of reactivation. Moreover, unaltered kinetics of reactivation against variation of temperature, pH, and duration of denaturation strongly suggests absence of proline cis/trans isomerization. Measurement of kinetics of (i) recovery of tertiary structure by protein fluorescence; (ii) incorporation of NAD from quantitation of bound cofactor; (iii) formation of substrate binding site by specific interaction with extrinsic fluorophore 1-anilino-8-naphthalene sulfonic acid and quenching by 5′-UMP, a competitive inhibitor; and (iv) recovery of activity indicate that they are all comparable. It appears that internal rearrangement of the protein during refolding, shielded from solvent, is the rate-limiting step of generation of cofactor binding site which ultimately leads to maturation of the holoenzyme structure.

Limited proteolysis and site-directed mutagenesis reveal the origin of microheterogeneity in Rhodotorula gracilis D-amino acid oxidase.

When analysed by isoelectric focusing, D-amino acid oxidase from the yeast Rhodotorula gracilis normally consists of three molecular isoforms (pI 7.8, 7.4 and 7.2, respectively) all with the same N-terminal sequence. However, only a single band of pI 7.8 is detected with the recombinant wild-type protein expressed in E. coli. To determine whether the molecular basis of this heterogeneity is due to proteolysed forms of the protein, we treated R. gracilis D-amino acid oxidase with various proteases. Limited proteolysis by chymotrypsin and thermolysin produced truncated and nicked monomeric holoenzymes containing two polypeptides of approximately 34 kDa (Met1-Leu312) and one of approximately 5 kDa (Ala319-Arg364 with chymotrypsin or Ala319-Ala362 with thermolysin). On the other hand, treatment with endoproteinase Glu-C gave a dimeric holoenzyme lacking the C-terminal SKL tripeptide. This cleavage of Glu365-Ser366 peptide bond caused the disappearance of the three isoelectric bands and a single homogeneous band (pI 7.2) appeared. To study this protein form, we used site-directed mutagenesis to produce a mutant form of R. gracilis D-amino acid oxidase lacking the SKL C-terminal tripeptide (which is the targeting sequence PTS1 for peroxisomal proteins). As expected, the SKL-deleted mutant gave a single band (pI 7.2) in isoelectric focusing. The three-band pattern of native yeast enzyme was generated by in vitro experiments using an equimolar mixture of the wild-type (pI 7.8) and the SKL-deleted recombinant (pI 7.2) DAAOs. The microheterogeneity of yeast DAAO thus stems from the association of two polypeptide chains differing in the C-terminal tripeptide, giving three different holoenzyme dimers.

Isolation and characterization of the d 1 domain of Pseudomonas aeruginosa nitrite reductase

Proteolitic digestion of nitrite reductase from Pseudomonas aeruginosa allows to obtain and purify a domain containing only the d 1 heme and constituted by two noncovalently bound peptides. This d 1 domain catalyzes oxygen consumption, and binds carbon monoxide with a kinetic constant slightly higher than the parental dimeric holoenzyme. The capacity to oxidize the physiological substrate, cytochrome c 551, is lost, even when the proteolytic c heme domain is added to this reaction mixture. This finding suggests that the two domains do not have a significant affinity for each other, and are kept together only by being part of the same polypeptide.

Characterization of Heme-deficient Neuronal Nitric-oxide Synthase Reveals a Role for Heme in Subunit Dimerization and Binding of the Amino Acid Substrate and Tetrahydrobiopterin

Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multifunctional oxidoreductase catalyzing oxidation of L-arginine to L-citrulline and NO, reduction of molecular oxygen to superoxide, and electron transfer to cytochromes. To investigate how binding of the prosthetic heme moiety is related to enzyme activities, cofactor, and L-arginine binding, as well as to secondary and quaternary protein structure, we have purified and characterized heme-deficient neuronal NO synthase. The heme-deficient enzyme, which had preserved its cytochrome c reductase activity, contained FAD and FMN, but virtually no tetrahydrobiopterin, and exhibited only marginal NO synthase activity. By means of gel filtration and static light scattering, we demonstrate that the heme-deficient enzyme is a monomer and provide evidence that heme is the sole prosthetic group controlling the quaternary structure of neuronal NO synthase. CD spectroscopy showed that most of the structural elements found in the dimeric holoenzyme were conserved in heme-deficient monomeric NO synthase. However, in spite of being properly folded, the heme-deficient enzyme did bind neither tetrahydrobiopterin nor the substrate analog N(G)-nitro-L-arginine. Our results demonstrate that the prosthetic heme group of neuronal NO synthase is requisite for dimerization of enzyme subunits and for the binding of amino acid substrate and tetrahydrobiopterin.

An Unusual Dehalogenating Peroxidase from the Marine Terebellid Polychaete Amphitrite ornata

The terebellid polychaete Amphitrite ornata produces no detectable volatile halogenated secondary metabolites, but frequently inhabits coastal marine sediments heavily contaminated with anthropogenic or biogenic haloaromatic compounds. This animal contains high levels of two very unusual enzymes, dehalogenating peroxidases. We have purified and partially characterized one of these dehaloperoxidases, DHP I. DHP I is a heme enzyme (Mr = 30,790) composed of two identical subunits (Mr = 15,529) and is very rich in the amino acids aspartic acid (+ asparagine) and glutamic acid (+ glutamine). The enzyme converts trihalogenated phenols, such as 2,4,6-tribromophenol, into dihalogenated quinones. The optimum pH for this reaction is 5.0. DHP I is also active against di- and monohalogenated phenols and will oxidize bromo-, chloro-, and fluorophenols. We have identified similar dehaloperoxidase activities in other infaunal polychaetes, including halometabolite-producing species.

Neurofilament-associated protein phosphatase 2A: Its possible role in preserving neurofilaments in filamentous states

Neurofilament phosphatase (NF-phosphatase) activity, which dephosphorylates NF proteins phosphorylated by cyclic AMP-dependent protein kinase (A-kinase), was detected in NF fractions prepared from bovine spinal cords. This phosphatase was suggested to be associated with NFs by gel filtration and sedimentation analysis and was further demonstrated by dephosphorylation-dependent binding assay of NFs to microtubules. The NF-associated NF-phosphatase was identified as a type of protein phosphatase 2A (PP2A) by (i) its complete inhibition with 100 nM okadaic acid, at which concentration the purified type 1 protein phosphatase (PP1) was inhibited only 25%; (ii) the absence of effect of inhibitor-2, a specific inhibitor of PP1, on the NF-phosphatase activity; and (iii) the detection of 38-kDa catalytic and 65-kDa regulatory subunits of PP2A by immunoblotting. The NF-associated PP2A was partially solubilized from NFs by a high concentration of MgSO4, and the solubilized PP2A was suggested by gel filtration to be a dimeric holoenzyme consisting of a 38-kDa catalytic and a 65-kDa regulatory subunit. Phosphorylated NF-L, which is assembly incompetent, was induced to assemble into filaments by dephosphorylation with PP2A. These results suggest a role of NF-associated PP2A in preserving filamentous forms of NF in neurons.

Regulation of protein kinase A subunits during germination of Mucor rouxii sporangiospores.

Levels of protein kinase A (PKA) subunits and of cAMP have been measured during aerobic germination of the sporangiospores of the dimorphic fungus Mucor rouxii; further, the holoenzyme and its catalytic (C) and regulatory (R) subunits have been visualized through sucrose gradient centrifugation. Sporangiospores contain around 0.06 microM of a dimeric holoenzyme species of 5.5 S and a sixfold excess of a free R subunit of 2.7 S. Both these species are proposed to be derived by proteolysis from the native forms. Enzymic activity at this stage is highly inhibited, as demonstrated with permeabilized cells. Immediately upon germination, and after a transient increase in cAMP concentration from 10 microM to 90 microM, C-subunit levels fall to 30%. After the onset of germination, the specific activity and concentration of both the 5.5 S holoenzyme species and the 2.7 S species of free R subunit decrease in parallel to the increase in total protein and volume. Net synthesis of C and R subunits to form a native holoenzyme species of 8.8 S is apparent 4 h onwards after germination. A significant increase in cellular concentration is observed at 6 h. At 7 h of growth, when germ-tube emission is complete, the holoenzyme concentration is around 0.23 microM; there is almost no free R subunit and the intracellular concentration of cAMP is around 3 microM. A role for PKA during germination and morphogenesis is discussed.

Analysis of subunit isoforms in protein phosphatase 2A holoenzymes from rabbit and Xenopus.

A dimeric and two trimeric forms of protein phosphatase 2A (PP2A) were purified from rabbit and Xenopus tissues and analyzed using antisera specific for the catalytic and regulatory subunits. The dimeric holoenzyme consists of a complex between a 36-kDa catalytic subunit associated with a approximately 65-kDa regulatory subunit. The two trimeric holoenzymes consist of the catalytic subunit complexed with 65- and 55-kDa subunits, or 65- and 72-kDa subunits. Antisera were raised against synthetic peptides specific for the alpha- and beta-isoforms of the 65-kDa (PR65 alpha/beta) and 55-kDa (PR55 alpha/beta) subunits identified by molecular cloning. Anti-peptide antisera to the 36-kDa catalytic subunit of PP2A were prepared against two selected regions: one specific for the alpha-isoform and one to a peptide common to both the alpha- and beta-isoforms. Immunochemical analysis of all three mammalian holoenzymes showed that the catalytic, 55- and 65-kDa subunits are both predominantly of the alpha-isoform, which is consistent with the peptide sequence data. The 65-kDa subunit of PP2A holoenzymes isolated from Xenopus skeletal muscle reacted with both anti-alpha and anti-beta PR65-specific antisera whereas the PP2A holoenzymes isolated from Xenopus oocytes reacted preferentially with the beta-specific antisera, indicating developmental changes in the expression of the 65-kDa subunit isoform. Taken together, these results show that the "core" subunits of the PP2A holoenzymes consist of the catalytic complexed with the 65-kDa subunit and that the association of the third subunit does not appear to be influenced by the isoform of these two core subunits.

Spatial arrangement of σ-factor and core enzyme of Escherichia coli RNA polymerase : A neutron solution scattering study

By means of neutron solution scattering we determined the position and orientation of core enzyme and σ-factor within the Escherichia coli RNA polymerase holoenzyme with the aim of improving existing models. The individual components, core enzyme (E) and σ-factor (σ), were highlighted by deuterium labeling and their center-to-center distances determined in the monomeric and the dimeric holoenzyme. The following distance parameters were obtained: d E 1 − σ 1 = 8.6(± 1) nm, d E 1 − E 2 = 11.5(± 1) nm, d σ 1 − σ 2 = 12.0(±0.7) nm, d E 1 − σ 2 = 9(± 3) nm. Using a triangulation procedure the position of the σ-factors, σ 1 and σ 2, were determined with respect to the mass center of the core enzyme molecules, E 1 and E 2 assuming a symmetrical arrangement of the holoenzyme molecules in the dimer ( C2 symmetry). In addition, the orientation of the σ-factor with respect to core enzyme was estimated by means of model calculations. The obtained model of holoenzyme depicts the σ-factor as buried in a groove of core enzyme, probably between the large subunits β′ and β.

On the FAD‐induced dimerization of apo‐lipoamide dehydrogenase from Azotobacter vinelandii and Pseudomonas fluorescens

The apoenzymes of lipoamide dehydrogenase from pig heart and from Pseudomonas fluorescens were prepared at pH 2.7 and pH 4.0, respectively, using a hydrophobic interaction chromatography procedure recently developed for lipoamide dehydrogenase from Azotobacter vinelandii and other flavoproteins [Van Berkel et al. (1988) Eur. J. Biochem. 178, 197-207]. The apoenzyme from pig heart, having 5% of residual activity, shows an equilibrium between the monomeric and dimeric species. Both the yield and the degree of reconstitution of dimeric holoenzyme is 75% of starting material under optimal conditions. The kinetics of reconstitution of pig heart apoenzyme differ slightly from that obtained with the apoenzyme prepared by acid ammonium sulfate precipitation at pH 1.5 [Kalse, J. F. and Veeger, C. (1968) Biochim. Biophys. Acta 159, 244-256]. The apoenzyme from P. fluorescens is in the monomeric state and shows negligible residual activity. The yield and degree of reconstitution of the dimeric holoenzyme is more than 90% of starting material. Reconstitution of the apoenzymes from A. vinelandii and P. fluorescens involves minimally a two-step sequential process. Initial flavin-binding results in regaining of full dichloroindophenol activity, quenching of tryptophan fluorescence and strong increase of FAD fluorescence polarization. In the second step, dimerization occurs as reflected by regain of lipoamide activity, strongly increased FAD fluorescence and increased hyperchroism of the visible absorption spectrum. The kinetics of FAD-induced dimerization are strongly dependent on the apoenzyme used. At 0 degrees C, the monomeric apoenzyme-FAD complex is either stabilized (P. fluorescens) or only transiently detectable (A. vinelandii). Dimerization of P. fluorescens enzyme is strongly stimulated in the presence of NADH.

DNA polymerase III holoenzyme of Escherichia coli. III. Distinctive processive polymerases reconstituted from purified subunits.

The 10 distinctive polypeptides of DNA polymerase III holoenzyme, purified as individual subunits or complexes, could be reconstituted to generate a polymerase with the high catalytic rate of the isolated intact holoenzyme. Functions and interactions of the subunits can be inferred from partial assemblies of the pol III core (alpha, epsilon, and theta subunits) with auxiliary subunits. The core possesses the polymerase and proofreading activities; the auxiliary subunits provide the core with processivity, the capacity to replicate long stretches of DNA without dissociating from the template. In a sequence of reconstruction steps, the beta subunit binds the primed template in an ATP-dependent manner through the catalytic action of a complex made up of the gamma, delta, delta', chi, and psi polypeptides. With the beta subunit in place, a processive polymerase is produced upon addition of the core. When the tau subunit is lacking, binding of polymerase to the primed template is less efficient and stable. The tau-less reconstituted polymerase is more prone to dissociation upon encountering secondary structures in the template in its path, such as a hairpin region in the single strand or a duplex region formed by a strand annealed to the template. With the tau subunit present, the interaction of the core.beta complex (the basic unit of a processive polymerase) with the primed template is strengthened. The tau-containing reconstituted polymerase can replicate DNA continuously through secondary structures in the template. The two distinctive kinds of processivity demonstrated by the tau-less and tau-containing reconstituted polymerases fit nicely into a scheme in which, organized as an asymmetric dimeric holoenzyme, the tau half is responsible for continuous synthesis of one strand, and the less stable half for discontinuous synthesis of the other.

Intrinsic luminescence studies on the apoenzyme and holoenzyme of lipoamide dehydrogenase.

The following fluorescence parameters have been investigated: emission and excitation spectra, relative quantum yields, polarization values and lifetimes of the apo‐ and holoenzyme of lipoamide dehydrogenase. Furthermore, fluorescence perturbation techniques have been used to characterize qualitatively the tryptophan and flavin environment in the protein. Also emission spectra at low temperature have been recorded to examine changes occurring on strong immobilization of the enzyme. From lifetimes and quantum yields of the protein fluorescence it can be concluded that the molecular structures of the two apoenzymes, which can be prepared from this enzyme, are different. The quenched protein fluorescence of the holoenzyme is characterized by a very short fluorescence lifetime due to radiationless energy transfer from tryptophan to flavin. Excitation polarization spectra of flavin and tryptophan fluorescences show that the chromophores are rigidly bound to the protein. Luminescence studies at liquid nitrogen temperatures reveal that the triplet state of the tryptophanyl‐residues is not involved in energy transfer to the flavin.Collisional fluorescence quenching with KI shows in the dimeric holoenzyme, in contrast with the free molecules, only a small reduction in fluorescence intensity of tryptophanyl and flavin residues. Charge effects might be responsible for these observations. It can be derived from the experimental results together with the indication that only one of the two tryptophans is transferring its excitation energy to the flavin, that the distance between donor and acceptor is 1.3–1.6 nm.