Macromolecular properties of cepacian in water and in dimethylsulfoxide

Cepacian is the exopolysaccharide produced by the majority of the so far investigated clinical strains of the Burkholderia cepacia complex. This is a group of nine closely related bacterial species that might cause serious lung infections in cystic fibrosis patients, in some cases leading to death. In this paper the aggregation ability and the conformational properties of cepacian chain were investigated to understand its role in biofilm formation. Viscosity and atomic force microscopy studies in water and in mixed (dimethylsulfoxide/water) solvent indicated the formation of double stranded molecular structures in aqueous solutions. Inter-residue short distances along cepacian chain were investigated by NOE NMR, which showed that two side chains of cepacian were not conformationally free due to strong interactions with the polymer backbone. These interactions were attributed to hydrogen bonding and contributed to structure rigidity.     

Synthesis of the catechols of natural and synthetic estrogens by using 2-iodoxybenzoic acid (IBX) as the oxidizing agent

A method for the synthesis of 2-hydroxyestrone/estradiol, 4-hydroxyestrone/estradiol, 3'-hydroxydiethylstilbestrol, 3'-hydroxyhexestrol, and 3'-hydroxydienestrol is reported, in which 2-iodoxybenzoic acid (IBX) and the corresponding phenolic estrogen are reacted. Treatment of the natural estrogens, estrone/estradiol, with stoichiometric amounts of IBX in dimethylformamide initially yielded a mixture of estrone/estradiol-2,3- and -3,4-quinones, which were reduced in situ to the corresponding catechols by treatment with a 1 M aqueous solution of ascorbic acid. Chromatographic separation of the reaction products afforded 2- and 4-hydroxyestrone/estradiol in good overall yields (79%). In the case of the synthetic estrogens containing two identical phenolic rings, protection of one ring is a prerequisite for the synthesis of the monocatechol. Thus, diethylstilbestrol and dienestrol were protected at one phenol ring as their methyl ethers. The resulting monophenols were treated with stoichiometric amounts of IBX for 1 h, followed by treatment with 1 M aqueous ascorbic acid to obtain the corresponding catechols in more than 70% yield. Furthermore, the catechol of diethylstilbestrol, protected at one ring, was reduced by catalytic hydrogenation at the C3-C4 double bond to obtain 3'-hydroxyhexestrol in 90% yield. Removal of the protected methoxy groups of the synthetic estrogen catechols was carried out by treatment with a 1 M solution of boron tribromide in dichloromethane. This method is highly efficient for the preparative scale synthesis of catechols of both natural and synthetic estrogens.     

Colonization of Aspergillus japonicus on synthetic materials and application to the production of fructooligosaccharides

The ability of Aspergillus japonicus ATCC 20236 to colonize different synthetic materials (polyurethane foam, stainless steel sponge, vegetal fiber, pumice stones, zeolites, and foam glass) and to produce fructooligosaccharides (FOS) from sucrose (165 g/L) is described. Cells were immobilized in situ by absorption, through direct contact with the carrier particles at the beginning of fermentation. Vegetal fiber was the best immobilization carrier as A. japonicus grew well on it (1.25 g/g carrier), producing 116.3 g/L FOS (56.3 g/L 1-kestose, 46.9 g/L 1-nystose, and 13.1 g/L 1-β-fructofuranosyl nystose) with 69% yield (78% based only in the consumed sucrose amount), giving also elevated activity of the β-fructofuranosidase enzyme (42.9 U/mL). In addition, no loss of material integrity, over a 2 day-period, was found. The fungus also immobilized well on stainless steel sponge (1.13 g/g carrier), but in lesser extents on polyurethane foam, zeolites, and pumice stones (0.48, 0.19, and 0.13 g/g carrier, respectively), while on foam glass no cell adhesion was observed. When compared with the FOS and β-fructofuranosidase production by free A. japonicus, the results achieved using cells immobilized on vegetal fiber were closely similar. It was thus concluded that A. japonicus immobilized on vegetal fiber is a potential alternative for high production of FOS at industrial scale.     

S100 proteins interact with the N-terminal domain of MDM2

S100 proteins interact with the transactivation domain and the C-terminus of p53. Further, S100B has been shown to interact with MDM2, a central negative regulator of p53. Here, we show that S100B bound directly to the folded N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and surface plasmon resonance experiments. This interaction with MDM2 (2-125) is a general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also interact with MDM2 (2-125). These interactions with S100 proteins do not result in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53 ubiquitylation in vitro.

Structured summary

MINT-7905256: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A6 (uniprotkb:P06703) by surface plasmon resonance (MI:0107)MINT-7905063: MDM2 (uniprotkb:Q00987) and s100A1 (uniprotkb:P23297) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905376: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) physically interact (MI:0915) by competition binding (MI:0405)MINT-7905130: s100A6 (uniprotkb:P06703) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905207: s100A6 (uniprotkb:P06703) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905043: s100B (uniprotkb:P04271) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905196: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905358: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) physically interact (MI:0915) by fluorescence polarization spectroscopy (MI:0053)MINT-7905220: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100B (uniprotkb:P04271) by surface plasmon resonance (MI:0107)MINT-7905104: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905229: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A1 (uniprotkb:P23297) by surface plasmon resonance (MI:0107)MINT-7905317, MINT-7905162: s100B (uniprotkb:P04271) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905238: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A2 (uniprotkb:P29034) by surface plasmon resonance (MI:0107)MINT-7905174, MINT-7905308: s100A1 (uniprotkb:P23297) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905247: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A4 (uniprotkb:P26447) by surface plasmon resonance (MI:0107)MINT-7905090: s100A2 (uniprotkb:P29034) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905142, MINT-7905326: MDM2 (uniprotkb:Q00987) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905185, MINT-7905347: s100A2 (uniprotkb:P29034) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)     

Magnetic and structural studies of novel tetranickel hydroxamates

The dinickel(II) compound [Ni₂(μ-OAc)₂(OAc)₂(μ-H₂O)(asy·dmen)₂]·2.5H₂O, 1; undergoes facile reaction in a 1:2 molar ratio with benzohydroxamic acid (BHA) in ethanol to give the novel nickel(II) tetranuclear hydroxamate complex [Ni₄(μ-OAc)₃(μ-BA)₃(asy·dmen)₃][OTf]₂·H₂O, 2, in which the bridging acetates, bridging two nickel atoms in 1, undergo a carboxylate shift from the μ₂-η¹:η¹ bridging mode of binding to the μ₃-η¹:η² bridging three nickel atoms in the tetramer. The structure of complex 2 was determined by single-crystal X-ray crystallography. The two monodentate acetates, water and two bidentate bridging acetates of two moles of complex 1 are replaced by three monodentate bridging acetates and three benzohydroxamates. Three nickel atoms in the tetramer, Ni(2), Ni(3) and Ni(4) are in a N₂O₄ octahedral environment, while the fourth nickel atom Ni(1) is in an O(6) octahedral environment. The Ni-Ni separations are Ni(1)-Ni(2) = 3.108 Å, Ni(1)-Ni(3) = 3.104 Å and Ni(1)-Ni(4) = 3.110 Å, which are longer than previously studied in dinuclear urease inhibited models but shorter than in the nickel(II) tetrameric glutarohydroxamate complex [Ni₄(μ-OAc)₂(μ-gluA₂)₂(tmen)₄][OTf]₂, isolated and characterized previously in this laboratory. Magnetic studies of the tetrameric complex show that the four Ni(II) ions are ferromagnetically coupled, leading to a total ground spin state S_T = 4. Three analogous tetranuclear nickel hydroxamates were prepared from AHA and BHA and the appropriate dinuclear complex with either sy·dmen or asy·dmen as capping ligands.     

Thermodynamic properties of BPTI variants with highly simplified amino acid sequences

We report the first detailed thermodynamic analysis of simplified proteins by differential scanning calorimetry (DSC). The experiments were carried out with five simplified BPTI variants, whose structures and activities have been reported, in which several residues not essential for specifying the tertiary structure were replaced by alanine. In most aspects, the thermodynamics of simplified proteins were very similar to, if not essentially identical with, those of natural proteins. In particular, they undergo a highly cooperative two-state thermal unfolding process with a large enthalpy change, which is a thermodynamic hallmark of the native state of natural globular proteins. Furthermore, the specific enthalpy and entropy changes upon unfolding at 110 degrees C were close to values invariably observed for small natural globular proteins (55 J g(-1) and ~16 J K(-1) g(-1), respectively). On the other hand, two simplified BPTI variants, BPTI-21 and BPTI-22 (containing 21 and 22 alanine residues), were enthalpically stabilized while entropically destabilized with respect to the reference BPTI-[5,55] molecule. This peculiar type of entropy-enthalpy compensation is in sharp contrast to the usual enthalpy destabilization/entropy stabilization observed in mutational studies of natural proteins. Overall, we conclude that a thermodynamic native state can be achieved by proteins encoded with extensively simplified sequences.     

Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii

Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q_A to Q_B at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q_B-niche. The results of competitive replacement with [¹⁴C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q_B-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.     

Fine structure of body wall cuticle of females of Meloidodera charis,Atalodera lonicerae,and Sarisodera hydrophila (Heteroderidae)

The body wall cuticle of adult females of Meloidodera charis, Atolodera lonicerae, and Sarisodera hydrophila is examined by transmission electron and light microscopy for comparison with Heterodera schachtii and previous observations of additional species of Heterodera, Globodera, and Punctodera. The cuticle of M. charis is least complex, consisting of layers A, B, C (with A outermost), and varies in overall thickness from 3 to 8 μm. As in other species, the cuticle is thickest in mature specimens. The cuticle of A. lonicerae is 6-9 μm thick; unlike M. charis it has an innermost layer, D, in addition to A, B, and C. The cuticle of S. hydrophila varies from 14 to 30 μm thick and includes a D layer similar to A. lonicerae; layer C is subdivided into additional zones relative to other heteroderids, and the external portion of the cuticle is infused with an electron-dense material. The presence of a D layer in A. lonicerae and S. hydrophila is a character state which is shared with Globodera spp. and Punctodera sp. The electron-dense material in the outer layers of S. hydrophila also occurs in Globodera spp. and Punctodera sp. On the other hand, H. schachtii resembles other Heterodera spp. as well as M. charis by the absence of a D layer and lack of electron-dense material in the outer layers. The pattern of occurrence of shared character states, including those of the cuticle, may be useful for phylogenetic analysis of Heteroderidae.     

Mutations in DIIS5 and the DIIS4-S5 linker of Drosophila melanogaster sodium channel define binding domains for pyrethroids and DDT

Mutations in the DIIS4-S5 linker and DIIS5 have identified hotspots of pyrethroid and DDT interaction with the Drosophila para sodium channel. Wild-type and mutant channels were expressed in Xenopus oocytes and subjected to voltage-clamp analysis. Substitutions L914I, M918T, L925I, T929I and C933A decreased deltamethrin potency, M918T, L925I and T929I decreased permethrin potency and T929I, L925I and I936V decreased fenfluthrin potency. DDT potency was unaffected by M918T, but abolished by T929I and reduced by L925I, L932F and I936V, suggesting that DIIS5 contains at least part of the DDT binding domain. The data support a computer model of pyrethroid and DDT binding.     

Evolutionary history of the Afro-Madagascan Ixora species (Rubiaceae): species diversification and distribution of key morphological traits inferred from dated molecular phylogenetic trees

Background and Aims

Previous work on the pantropical genus Ixora has revealed an Afro-Madagascan clade, but as yet no study has focused in detail on the evolutionary history and morphological trends in this group. Here the evolutionary history of Afro-Madagascan Ixora spp. (a clade of approx. 80 taxa) is investigated and the phylogenetic trees compared with several key morphological traits in taxa occurring in Madagascar.

Methods

Phylogenetic relationships of Afro-Madagascan Ixora are assessed using sequence data from four plastid regions (petD, rps16, rpoB-trnC and trnL-trnF) and nuclear ribosomal external transcribed spacer (ETS) and internal transcribed spacer (ITS) regions. The phylogenetic distribution of key morphological characters is assessed. Bayesian inference (implemented in BEAST) is used to estimate the temporal origin of Ixora based on fossil evidence.

Key Results

Two separate lineages of Madagascan taxa are recovered, one of which is nested in a group of East African taxa. Divergence in Ixora is estimated to have commenced during the mid Miocene, with extensive cladogenesis occurring in the Afro-Madagascan clade during the Pliocene onwards.

Conclusions

Both lineages of Madagascan Ixora exhibit morphological innovations that are rare throughout the rest of the genus, including a trend towards pauciflorous inflorescences and a trend towards extreme corolla tube length, suggesting that the same ecological and selective pressures are acting upon taxa from both Madagascan lineages. Novel ecological opportunities resulting from climate-induced habitat fragmentation and corolla tube length diversification are likely to have facilitated species radiation on Madagascar.     

A structural basis for the pH-dependent increase in fluorescence efficiency of chromoproteins

Within the fluorescent protein and chromoprotein family, the phenomenon of photoswitching is both intriguing and biotechnologically useful. Illumination of particular chromoproteins with intense light results in dramatic increases in fluorescence efficiency (termed kindling) and involves cis-trans isomerization of the chromophore. Here we report that chromophore isomerization can also be driven via alteration in pH. Specifically, we demonstrate that a number of naturally occurring chromoproteins, and their engineered variants, undergo a dramatic 20-100-fold increase in fluorescence efficiency at alkaline pH (>pH9.0). We have determined to 1.8 A resolution the structure of one such chromoprotein, Rtms5(H146S), in its highly far-red fluorescent form (Phi(F), 0.11 at pH 10.7) and compared it to the structure of the non-fluorescent form (Phi(F), 0.002 at pH 8.0). At high pH, the cyclic tri-peptide chromophore was observed to be mobile and distributed between a trans non-coplanar and a cis coplanar conformation, whereas at the lower pH, only a trans non-coplanar chromophore was observed. Calculation of pK(a) values suggested that titration of the side-chain of the conserved Glu215 close to the chromophore is involved in promoting the cis-coplanar conformation. Collectively, our data establish that isomerization to form a coplanar chromophore is a basis of the increased fluorescence efficiency at high pH. The phenomenon of pH-induced fluorescence gain has similarities with photoswitching, thereby providing a model to study the mechanism of kindling.     

Low molecular weight chitosans—Preparation with the aid of pronase,characterization and their bactericidal activity towards Bacillus cereus and Escherichia coli

The homogeneous low molecular weight chitosans (LMWC) of molecular weight 9.5–8.5 kDa, obtained by pronase catalyzed non-specific depolymerization (at pH 3.5, 37 °C) of chitosan showed lyses of Bacillus cereus and Escherichia coli more efficiently (100%) than native chitosan (< 50%). IR and ¹H-NMR data showed decrease in the degree of acetylation (14–19%) in LMWC compared to native chitosan (∼ 26%). Minimum inhibitory concentration of LMWC towards 10⁶ CFU ml^− 1 of B. cereus was 0.01% (w/v) compared to 0.03% for 10⁴ CFU ml^− 1 of E. coli. SEM revealed pore formation as well as permeabilization of the bacterial cells, as also evidenced by increased carbohydrate and protein contents as well as the cytoplasmic enzymes in the cell-free supernatants. N-terminal sequence analyses of the released proteins revealed them to be cytoplasmic/membrane proteins. Upon GLC, the supernatant showed characteristic fatty acid profiles in E. coli, thus subscribing to detachment of lipopolysaccharides into the medium, whereas that of B. cereus indicated release of surface lipids. The mechanism for the observed bactericidal activity of LMWC towards both Gram-positive and Gram-negative bacteria has been discussed.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Optimized variants of the cold shock protein from in vitro selection: structural basis of their high thermostability

The bacterial cold shock proteins (Csp) are widely used as models for the experimental and computational analysis of protein stability. In a previous study, in vitro evolution was employed to identify strongly stabilizing mutations in Bs-CspB from Bacillus subtilis. The best variant found by this approach contained the mutations M1R, E3K and K65I, which raised the midpoint of thermal unfolding of Bs-CspB from 53.8 degrees C to 83.7 degrees C, and increased the Gibbs free energy of stabilization by 20.9 kJ mol(-1). Another selected variant with the two mutations A46K and S48R was stabilized by 11.1 kJ mol(-1). To elucidate the molecular basis of these stabilizations, we determined the crystal structures of these two Bs-CspB variants. The mutated residues are generally well ordered and provide additional stabilizing interactions, such as charge interactions, additional hydrogen bonds and improved side-chain packing. Several mutations improve the electrostatic interactions, either by the removal of unfavorable charges (E3K) or by compensating their destabilizing interactions (A46K, S48R). The stabilizing mutations are clustered at a contiguous surface area of Bs-CspB, which apparently is critically important for the stability of the beta-barrel structure but not well optimized in the wild-type protein.     

Isolation, characterization and structural studies of amorpha - 4, 11-diene synthase (ADS(3963)) from Artemisia annua L

With the escalating prevalence of malaria in recent years, artemisinin demand has placed considerable stress on its production worldwide. At present, the relative low­yield of artemisinin (0.01­1.1 %) in the source plant (Artemisia annua L. plant) has imposed a serious limitation in commercializing the drug. Amorpha­4, 11­diene synthase (ADS) has been reported a key enzyme in enhancing the artemisinin level in Artemisia annua L. An understanding of the structural and functional correlations of Amorpha­4, 11­diene synthase (ADS) may therefore, help in the molecular up­regulation of the enzyme. In this context, an in silico approach was used to study the ADS₃₉₆₃ (3963 bp) gene cloned by us, from high artemisinin (0.7­0.9% dry wt basis) yielding strain of A. annua L. The full­length putative gene of ADS₃₉₆₃ was found to encode a protein consisting of 533 amino acid residues with conserved aspartate rich domain. The isoelectric point (pI) and molecular weight of the protein were 5.25 and 62.2 kDa, respectively. The phylogenetic analysis of ADS genes from various species revealed evolutionary conservation. Homology modeling method was used for prediction of the 3D structure of ADS3963 protein and Autodock 4.0 version was used to study the ligand binding. The predicted 3D model and docking studies may further be used in characterizing the protein in wet laboratory.     

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.     

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.     

Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly

Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.     

Molecular properties of the glucosaminidase AcmA from Lactococcus lactis MG1363: Mutational and biochemical analyses

The major autolysin AcmA of Lactococcus lactis ssp. cremoris MG1363 is a modular protein consisting of an N-terminal signal sequence, a central enzymatic region (glu_acma as a glucosaminidase), and a C-terminal cell-recognition domain (LysM123). glu_acma (about 160 amino acids) belongs to the glycoside hydrolase (GH) 73 family, and the two acidic residues E128 and D153 have been thought to be catalytically important. In this study, amino-acid substitution analysis of AcmA was first carried out in the Escherichia coli system. Point mutations E94A, E94Q, E128A, D153A, and Y191A markedly reduced cell-lytic activity (3.8%, 1.1%, 4.2%, 4.8%, and 2.4%, respectively), whereas E128Q and D153N retained significant residual activities (32.1% and 44.0%, respectively). On the other hand, Y191F and Y191W mutations retained high activities (66.2% and 46.0%, respectively). These results showed that E94 (rather than E128 and D153) and the aromatic residue Y191 probably play important roles in catalysis of AcmA. Together with mutational analysis of another GH73 glucoaminidase Glu_atlwm from the Staphylococcus warneri M autolysin Atl_WM, these results suggested that the GH73 members cleave a glycosidic bond via a substrate-assisted mechanism, as postulated in the GH20 members. AcmA and Glu_atlwm were purified from E. coli recombinant cells, and their enzymatic properties were studied.