Cleavage of peptide bonds bearing ionizable amino acids at P1 by serine proteases with hydrophobic S1 pocket

Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, and ∼10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S₁ pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.     

Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (FoxLOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-²H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the ²H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser³⁴⁸ in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser³⁴⁸, binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.     

Complete genome sequence of the termite hindgut bacterium Spirochaeta coccoides type strain (SPN1(T)), reclassification in the genus Sphaerochaeta as Sphaerochaeta coccoides comb. nov. and emendations of the family Spirochaetaceae and the genus Sphaerochaeta

Spirochaeta coccoides Dröge et al. 2006 is a member of the genus Spirochaeta Ehrenberg 1835, one of the oldest named genera within the Bacteria. S. coccoides is an obligately anaerobic, Gram-negative, non-motile, spherical bacterium that was isolated from the hindgut contents of the termite Neotermes castaneus. The species is of interest because it may play an important role in the digestion of breakdown products from cellulose and hemicellulose in the termite gut. Here we provide a taxonomic re-evaluation for strain SPN1^T, and based on physiological and genomic characteristics, we propose its reclassification as a novel species in the genus Sphaerochaeta, a recently published sister group of the Spirochaeta. The 2,227,296 bp long genome of strain SPN1^T with its 1,866 protein-coding and 58 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Research of the (E/Z)-isomerization of carotenoids in Pécs since the 1970s

Geometrical configuration of the polyene chain of approximately 40 mono- and di-cis carotenoids was determined from 1970 through 1990. Subsequently, the kinetic, equilibrium and thermodynamic parameters (k, K, A, E_A, ΔH^#, ΔG^#, ΔS^#) of the reversible thermal isomerization of several symmetrical and unsymmetrical carotenoids were calculated. The rate of the iodine-catalyzed photoisomerization of (all-E)-, (9Z)- and (13Z)-zeaxanthin was compared and the ‘specific rate’ (per unit light energy at given wavelengths) of the iodine-catalyzed photoisomerization for several (13Z)-carotenoids was investigated. As the missing links of the biosynthetic pathway of paprika-carotenoids, carotenoids containing new end groups were isolated; their sterically unhindered mono-cis isomers were also prepared and their geometrical configuration was determined. The investigation concentrated on the substrate specificity of the enzyme violaxanthin-deepoxidase, the light-induced formation of (13Z)-violaxanthin in green leaves, the binding of xanthophylls to the bulk light-harvesting complex (LHC) of photosystem II in higher plants, the biochemical basis of color as an aesthetic quality in Citrus-fruits and the (9Z)-epoxycarotenoid cleavage enzyme activity for ABA biosynthesis. Recently (9Z)-capsanthin-5,6-epoxide and capsoneoxanthin, two novel carotenoids have been isolated from natural sources.     

Detection of divinyl ether synthase in Lily-of-the-Valley (Convallaria majalis) roots

Incubations of linoleic acid with cell-free preparations from Lily-of-the-Valley (Convallaria majalis L., Ruscaceae) roots revealed the presence of 13-lipoxygenase and divinyl ether synthase (DES) activities. Exogenous linoleic acid was metabolized predominantly into (9Z,11E,1′E)-12-(1′-hexenyloxy)-9,11-dodecadienoic (etheroleic) acid. Its identification was confirmed by the data of ultraviolet spectroscopy, mass spectra, ¹H NMR, COSY, catalytic hydrogenation. The isomeric divinyl ether (8E,1′E,3′Z)-12-(1′,3′-nonadienyloxy)-8-nonenoic (colneleic) acid was detected as a minor product. Incubations with linoleic acid hydroperoxides revealed that 13-hydroperoxide was a preferential substrate, while the 9-hydroperoxide was utilized with lesser efficiency.     

Photobacterium marinum sp. nov., a marine bacterium isolated from a sediment sample from Palk Bay,India

The novel, cream colored, Gram-staining-negative, rod-shaped, motile bacteria, designated strains AK15^T and AK18, were isolated from sediment samples collected from Palk Bay, India. Both strains were positive for arginine dihydrolase, lysine decarboxylase, oxidase, nitrate reduction and methyl red test. The major fatty acids were C_16:0, C_{18:1 ω7c}, C_{16:1 ω7c} and/or C_{16:1 ω6c} and/or iso-C_15:0 2-OH (summed feature 3). Polar lipids content of strains AK15^T and AK18 were found to bephosphatidylethanolamine (PE), two unidentified phospholipids (PL1 and PL2) and three unidentified lipids (L1-L3). The 16S rRNA gene sequence analysis indicated strains AK15^T and AK18 as the members of the genus Photobacterium and closely related to the type strain Photobacterium jeanii with pair-wise sequence similarity of 96.7%. DNA–DNA hybridization between strain AK15^T and AK18 showed a relatedness of 87%. Based on data from the current polyphasic study, strains AK15^T and AK18 are proposed as novel species of the genus Photobacterium, for which the name Photobacterium marinum sp. nov. is proposed. The type strain of Photobacterium marinum is AK15^T (=MTCC 11066^T = DSM 25368^T).     

Enhancement of YD spin relaxation by the CaMn4 cluster in photosystem II detected at room temperature: A new probe for the S-cycle

The long-lived, light-induced radical Y_D^• of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn₄ cluster, situated ∼ 30 Å away. In this study we report a transient step-change increase in Y_D^• EPR intensity upon the application of a single laser flash to S₁ state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of Y_D^• by the OEC in the S₂ state, as a consequence of the single laser flash turnover. The post-flash decay reflected S₂ → S₁ back-turnover, as confirmed by their correlations with independent measurements of S₂ multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using Y_D^• as a probe.     

Base-pair exchange kinetics of the imino and amino protons of the 3′-phenazinium tethered DNA-RNA duplex,r(5′GAUUGAA3′):d(5′TCAATC3′-Pzn), and their comparison with those of B-DNA duplex

The dynamics of the opening-closing of the constituent base-pairs as well as of the exchange kinetics of the base-paired imino and amino protons with water in a DNA-RNA hybrid, [^5′r(G¹A²U³U⁴G⁵A⁶A⁷)^3′]:^5′p[d(T⁸C⁹A¹⁰A¹¹T¹²C¹³)]^3′-Pzn] duplex (I), are reported here in details for the first time. The exchange kinetics of amino and imino protons in the DNA-RNA hybrid (duplex I) have been compared with identical studies on the following B-DNA duplexes: d(C¹G²T³A⁴C⁵G⁶)₂ (II), d[p(^5′T¹G²T³T⁴T⁵G⁶ G⁷C⁸)^3′]:d[p(^5′C⁹C¹⁰A¹¹A¹²A¹³C¹⁴A¹⁵)^3′] (III), d(C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶)₂ (IV) and d(C¹G²C³G⁴C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶C¹⁷G¹⁸C¹⁹G²⁰)₂ (V). This comparative study shows that the life-times τ_o of various base-pairs in the DNA-RNA hybrid (I) varies in the range of ∼ 1 ms, and they are quite comparable to those of the shorter B-DNA duplexes (II) and (III), but very different from the τ_o of the larger duplexes (IV) and (V): the τ_o for the base pair of T¹¹ and T¹² residues in the 20-mer (duplex V) are 2.9 ± 2.3 ms and 23.2 ± 8.9 ms, respectively, while the corresponding τ_o in the 12-mer (duplex IV) are 2.8 ± 2.2 ms and 17.4 ± 5.4 ms. It has also been shown that the total energy of activation (E_a) assessed from the exchange rates of both imino and amino protons, representing energetic contributions from both base-pair and helix opening-closing as well as from the exchange process of the imino protons from the open state with the bound water, is close to the E_a of the short B-DNA duplex (E_a ≈ 28–47 kcal/mol).     

Dark recovery of the Chl a fluorescence transient (OJIP) after light adaptation: The qT-component of non-photochemical quenching is related to an activated photosystem I acceptor side

The dark recovery kinetics of the Chl a fluorescence transient (OJIP) after 15 min light adaptation were studied and interpreted with the help of simultaneously measured 820 nm transmission. The kinetics of the changes in the shape of the OJIP transient were related to the kinetics of the qE and qT components of non-photochemical quenching. The dark-relaxation of the qE coincided with a general increase of the fluorescence yield. Light adaptation caused the disappearance of the IP-phase (20-200 ms) of the OJIP-transient. The qT correlated with the recovery of the IP-phase and with a recovery of the re-reduction of P700⁺ and oxidized plastocyanin in the 20-200 ms time-range as derived from 820 nm transmission measurements. On the basis of these observations, the qT is interpreted to represent the inactivation kinetics of ferredoxin-NADP⁺-reductase (FNR). The activation state of FNR affects the fluorescence yield via its effect on the electron flow. The qT therefore represents a form of photochemical quenching. Increasing the light intensity of the probe pulse from 1800 to 15000 μmol photons m⁻² s⁻¹ did not qualitatively change the results. The presented observations imply that in light-adapted leaves, it is not possible to ‘close’ all reaction centers with a strong light pulse. This supports the hypothesis that in addition to Q_A a second modulator of the fluorescence yield located on the acceptor side of photosystem II (e.g., the occupancy of the Q_B-site) is needed to explain these results. Besides, some of our results indicate that in pea leaves state 2 to 1 transitions may contribute to the qI-phase.     

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T_m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E_a) was found to be 44.0 ± 0.3 kcal mol⁻¹. The activation enthalpy (ΔH^∗), free energy change (ΔG^∗) and entropy (ΔS^∗) were estimated to be 43 ± 4 kcal mol⁻¹, −26 ± 3 kcal mol⁻¹ and 204 ± 10 cal mol⁻¹ K⁻¹, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P₁ position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.     

Mycoheterotrophy evolved from mixotrophic ancestors: evidence in Cymbidium (Orchidaceae)

Background and Aims

Nutritional changes associated with the evolution of achlorophyllous, mycoheterotrophic plants have not previously been inferred with robust phylogenetic hypotheses. Variations in heterotrophy in accordance with the evolution of leaflessness were examined using a chlorophyllous–achlorophyllous species pair in Cymbidium (Orchidaceae), within a well studied phylogenetic background.

Methods

To estimate the level of mycoheterotrophy in chlorophyllous and achlorophyllous Cymbidium, natural ¹³C and ¹⁵N contents (a proxy for the level of heterotrophy) were measured in four Cymbidium species and co-existing autotrophic and mycoheterotrophic plants and ectomycorrhizal fungi from two Japanese sites.

Key Results

δ¹³C and δ¹⁵N values of the achlorophyllous C. macrorhizon and C. aberrans indicated that they are full mycoheterotrophs. δ¹³C and δ¹⁵N values of the chlorophyllous C. lancifolium and C. goeringii were intermediate between those of reference autotrophic and mycoheterotrophic plants; thus, they probably gain 30–50 % of their carbon resources from fungi. These data suggest that some chlorophyllous Cymbidium exhibit partial mycoheterotrophy (= mixotrophy).

Conclusions

It is demonstrated for the first time that mycoheterotrophy evolved after the establishment of mixotrophy rather than through direct shifts from autotrophy to mycoheterotrophy. This may be one of the principal patterns in the evolution of mycoheterotrophy. The results also suggest that the establishment of symbiosis with ectomycorrhizal fungi in the lineage leading to mixotrophic Cymbidium served as pre-adaptation to the evolution of the mycoheterotrophic species. Similar processes of nutritional innovations probably occurred in several independent orchid groups, allowing niche expansion and radiation in Orchidaceae, probably the largest plant family.     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and YZ radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S₁ and S₃ states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11–15 signal, were detected in Ca²⁺-depleted PS II. The g=11–15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S₂ in Ca²⁺-depleted PS II. The singlet-like signal was associated with the g=11–15 signal but not with the Y_Z (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y_Z radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y_Z radical was discussed.     

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E

A critical point in the V₁ sector and entire V₁V_O complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G_1–59) of the V₁V_O ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra of G_1–59 in the absence and presence of the N-terminal peptides E_1–18 and E_18–38 as well as the produced and purified C-terminal segment (E_39–233) shows specific interactions only with the peptide fragment E_18–38. The binding of this peptide occurs via the residues M₁, V₂, S₃, and K₅ as well for V₂₂, S₂₃, K₂₄, A₂₅ and R₂₆ of G_1–59. The specific E_18–38/G_1–59 binding has been confirmed by fluorescence correlation spectroscopy data. The E_18–38 peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E_18–38/G_1–59 complex reveals the orientation of E_18–38 relative to G_1–59 via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.     

Full characterization of PDX, a neuroprotectin/protectin D1 isomer, which inhibits blood platelet aggregation

Our study aimed to establish the complete structure of the main dihydroxy conjugated triene issued from the lipoxygenation (soybean enzyme) of docosahexaenoic acid, named PDX, an isomer of protectin/neuroprotectin D1 (PD1/NPD1) described by Bazan and Serhan. NMR approaches and other chemical characterization (e.g. GC-MS, HPLC and LC-MS/MS) indicated that PDX is 10(S),17(S)-dihydroxy-docosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid. The use of ¹⁸O₂ and mass spectrometry showed that PDX is a double lipoxygenation product. Its structure differs from PD1, with E,Z,E geometry (PDX) instead of E,E,Z (PD1) and S configuration at carbon 10 instead of R. PDX inhibits human blood platelet aggregation at sub-micromolar concentrations.     

Analysis of PDE6D and PDE6G genes for generalised progressive retinal atrophy (gPRA) mutations in dogs

The δ and γ subunits of the cGMP-phosphodiesterase (PDE6D, PDE6G) genes were screened in order to identify mutations causing generalised progressive retinal atrophy (gPRA) in dogs. In the PDE6D gene, single nucleotide polymorphisms (SNP) were observed in exon 4, in introns 2 and 3 and in the 3'' untranslated region (UTR) of different dog breeds. In the coding region of the PDE6G gene, exclusively healthy Labrador Retrievers showed an A → G transition in exon 4 without amino acid exchange. SNP were also observed in introns 1 and 2 in different dog breeds. The different SNP were used as intragenic markers to investigate the involvement of both genes in gPRA. The informative substitutions allowed us to exclude mutations in the PDE6D and PDE6G genes as causing retinal degeneration in 15 of the 22 dog breeds with presumed autosomal recessively transmitted (ar) gPRA.     

Cytotoxic trans-oriented iminoether platinum complexes - Kinetics of binding to DNA oligonucleotides determined by N NMR spectroscopy

The kinetics of reactions between cytotoxic trans-oriented iminoether platinum complexes and DNA oligonucleotides have been studied by 1D and 2D [¹H, ¹⁵N] HMQC NMR spectroscopy. The results for the two isomers of the mono-iminoether compound trans-[PtCl₂(NH₃){E/Z-HNC(OMe)Me}] (trans-E and trans-Z) are compared with those of the bis-iminoether derivative trans-[PtCl₂{E-HNC(OMe)Me}₂] (trans-EE). Earlier we have shown that quite unexpectedly, trans-EE is practically inert towards a central GG residue in a 12-mer double-helical duplex. We now show that the less bulky trans-E and trans-Z compounds do bind to the interior of the duplex [5′-d(G₁G₂T₃A₄C₅C₆G₇G₈ T₉A₁₀C₁₁C₁₂)]₂ which contains terminal and central “hot” GG site. The platination by trans-E and trans-Z is as expected most pronounced for the solvent exposed, terminal GG-step but significantly, competitive binding is also observed for the central GG-step. The rate of platination of the terminal G-sites is almost an order of magnitude larger for the oligomer than for the monomer GMP which was studied for comparison. The role of trans-platinum carrier ligands in influencing the type and rate of formation of adducts with DNA and other relevant biomolecules is discussed.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

Glucogalactan: A polysaccharide isolated from the cell-wall of Verticillium Lecanii

A water-soluble polysaccharide was extracted with alkali from the cell wall of Verticillium lecanii (also called Lecanicillium lecanii). After freezing and thawing, the water-soluble fraction was purified by gel filtration chromatography on Sepharose CL-6B and eluted as one peak by HPSEC/RID. Monosaccharide analysis showed galactose and glucose (1.1:1), with traces of mannose (<1%). The structural characteristics were determined by spectroscopic analysis, FT-IR and 1D and 2D ¹H and ¹³C NMR, and methylation results. On the basis of the data obtained, the following structure of the polysaccharide (E₃S_IV fraction) was established: