Acidic Substrates Research Articles

Development of a Novel Method for Feeding a Mixture of L-Lactic Acid and Acetic Acid in Fed-Batch Culture of Ralstonia eutropha for Poly-D-3-Hydroxybutyrate Production.

Fermentative production of poly-D-3-hydroxybutyrate [P(3HB)] from a mixture of L-lactic acid and acetic acid by Ralstonia eutropha was investigated. For fed-batch culture with cell density, it is necessary to control the concentration of these organic acids in the culture medium below the inhibitory level for cell growth. Therefore, a novel feeding method, termed the computer-controlled pH-stat substrate feeding method, was developed using the rate of increase of the pH (pH-increasing rate) of the culture medium as an indicator for feed control. The pH-increasing rate, which was calculated every minute by a pH meter-linked computer, represented secondary information regarding substrate consumption by cells. When the pH-increasing rate decreased to 5% of the maximum increasing rate, acidic substrate solution was fed into the fermentor until the pH was reduced to 7.00. Using this feeding strategy, the cell concentration and PHA content obtained in 42 h were 75.0 g/l and 73.1% (w/w), respectively, resulting in a high P(3HB) productivity of 1.30 g/l.h.

Black Rats, Island Characteristics, and Colonial Nesting Birds in the Mediterranean: Consequences of an Ancient Introduction

Abstract: The devastation of island faunas by alien species has been instrumental in raising concerns about the global threat to biological diversity. Colonial nesting species, often restricted to islands, have been affected severely. Eradication of introduced species as a means to alleviate the problem is usually done with little or no understanding of the mechanisms governing interactions between introduced and native species. Such an understanding could help target management action. We analyzed how island area, rock substrate, bird species biology, and presence of an introduced species, the black rat ( Rattus rattus), interact to explain the distribution and abundance of colonial nesting birds on a set of 72 islands from six archipelagos in the western Mediterranean. Rats were introduced to this region over 2000 years ago, and these communities have had time to reach an equilibrium. Using general linear models, we show that rats have affected species distributions more on the smaller islands and on islands with an acidic or neutral rock substrate; larger bird species are more resistant. On the smaller islands, where rat densities are highest, larger colonial birds are negatively affected. On larger islands, where rat densities are lower and fluctuate form year to year, larger colonial birds can maintain healthy populations despite the presence of rats. Although rats caused local extinction or reduction in bird abundance, the presence of islands varying in size and/or substrate allowed most archipelagos to retain their suite of colonial nesting bird species, despite a presumably reduced abundance for most species.

Evaluation of arsenite sorption in Spanish soils

Arsenite sorption was studied at different temperatures (30,40, and 50°C) to investigate processes that remove arsenite from soil solution (adsorption or precipitation), and if adsorption was taking place, how many sites were involved in this process, and their nature. Adsorption was the only reaction reducing levels of soluble arsenite in the two alkaline soils used in this experiment, Jijona and Agost; however, arsenite precipitation occurred in an acidic substrate (pH 4.0), Galicia soil. Iron (Fe) oxides and clay minerals were the soil components controlling arsenite sorption in the Jijona soil (pH 7.9 and high levels of Fe oxides). Calcite and clay minerals were the inorganic constituents involved in arsenite sorption in the Agost soil (pH 8.0 and high levels of CaCO3). Arsenite sorption was an endothermic and non‐spontaneous process. The fact that the higher the temperature, the higher the arsenite sorption, however, was likely due to an increase in the diffusion rate.

Trends in the adsorption of mono-end-capped polystyrenes onto polar substrates: Theoretical predictions and experimental observations

Predictions of the influence of the solvent quality (X), the segment-substrate interaction parameter (xps), the chain length (N), and the end cap-substrate interaction parameter (A) on the equilibrium-adsorbed amount from a dilute solution of end-functionalized homopolymers (r) onto a solid substrate, when both the end cap and polymer segments compete for sites on the surface, were made through application of the self-consistent-field (SCF) theory. These predictions were tested and evaluated through measurement of the equilibrium-adsorbed amount, from a dilute solution of single-end-functionalized custom synthesized relatively monodisperse polystyrenes, possessing an end group (X) chosen to interact with a mildly acidic inorganic substrate (Aerosil 130) and a mildly basic inorganic substrate (aluminum oxide C) through dipole-dipole interactions, hydrogen-bonding interactions, and specific acid-base interactions, as a function of X, Xps, Δ, and N. A protocol was developed for the numerical estimation of Δ through the establishment of dimensionless ratios of the type Γ X /Γ H , by experiment and through SCF calculation, as a function of A, for polymer/solvent/ substrate systems characterized by specific combinations of X, Xps, and N.

Mutation of recombinant catalytic subunit alpha of the protein kinase CK2 that affects catalytic efficiency and specificity.

In order to understand better the structural and functional relations between protein kinase CK2 catalytic subunit, the triphosphate moiety of ATP, the catalytic metal and the peptidic substrate, we built a structural model of Yarrowia lipolytica protein kinase CK2 catalytic subunit using the recently solved three-dimensional structure of the maize enzyme and the structure of cAMP-dependent protein kinase peptidic inhibitor (1CDK) as templates. The overall structure of the catalytic subunit is close to the structure solved by Niefind et al. It comprises two lobes, which move relative to each other. The peptide used as substrate is tightly bound to the enzyme, at specific locations. Molecular dynamic calculations in combination with the study of the structural model led us to identify amino acid residues close to the triphosphate moiety of ATP and a residue sufficiently far from the peptide that could be mutated so as to modify the specificity of the enzyme. Site-directed mutagenesis was used to replace by charged residues both glycine-48, a residue located within the glycine-rich loop, involved in binding of ATP phosphate moiety, and glycine-177, a residue close to the active site. Kinetic properties of purified wild-type and mutated subunits were studied with respect to ATP, MgCl(2) and protein kinase CK2 specific peptide substrates. The catalytic efficiency of the G48D mutant increased by factors of 4 for ATP and 17.5 for the RRRADDSDDDDD peptide. The mutant G48K had a low activity with ATP and no detectable activity with peptide substrates and was also inhibited by magnesium. An increased velocity of ADP release by G48D and the building of an electrostatic barrier between ATP and the peptidic substrate in G48K could explain these results. The kinetic properties of the mutant G177K with ATP were not affected, but the catalytic efficiency for the RRRADDSDDDDD substrate increased sixfold. Lysine 177 could interact with the lysine-rich cluster involved in the specificity of protein kinase CK2 towards acidic substrate, thereby increasing its activity.

Vascular plant species richness in Alaskan arctic tundra: the importance of soil pH

Summary 1 Species diversity in the Arctic varies dramatically across abiotic gradients, including topography, moisture, pH and nutrient availability. We hypothesized that vascular plant species density, richness and diversity in Alaskan tundra would be correlated with: (i) site age, (ii) soil pH, (iii) above‐ground productivity and biomass, and (iv) spatial heterogeneity. We sampled moist tussock, snowbed and watertrack communities in three sites that differed in substrate age (11 000–4.5 million years since deglaciation) for a variety of environmental and vegetation data over one growing season. 2 Productivity, biomass and heterogeneity were not consistently correlated with species density. However, variation in canopy height was correlated with species density and richness in a unimodal fashion, suggesting that heterogeneity of the light regime may be important for maintaining higher species numbers. 3 The 11 000‐year‐old site supported more vascular plant species than the two older sites, primarily due to greater numbers of forb species on the youngest site. 4 Soil pH was significantly positively correlated with species richness (R2 = 0.82) and species density (R2 = 0.61). In general the species found on acidic substrates (pH < 5.5) also occurred on non‐acidic substrates (pH > 5.5). 5 This pattern of higher richness with higher pH occurs across other tundra types throughout the Arctic, suggesting that soil pH is an important filter of the regional species pool within northern regions, although other factors may become more important at local scales.

The Molecular Structure of Hyperthermostable Aromatic Aminotransferase with Novel Substrate Specificity from Pyrococcus horikoshii

Aromatic amino acid aminotransferase (ArATPh), which has a melting temperature of 120 degrees C, is one of the most thermostable aminotransferases yet to be discovered. The crystal structure of this aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii was determined to a resolution of 2.1 A. ArATPh has a homodimer structure in which each subunit is composed of two domains, in a manner similar to other well characterized aminotransferases. By the least square fit after superposing on a mesophilic ArAT, the ArATPh molecule exhibits a large deviation of the main chain coordinates, three shortened alpha-helices, an elongated loop connecting two domains, and a long loop transformed from an alpha-helix, which are all factors that are likely to contribute to its hyperthermostability. The pyridine ring of the cofactor pyridoxal 5'-phosphate covalently binding to Lys(233) is stacked parallel to F121 on one side and interacts with the geminal dimethyl-CH/pi groups of Val(201) on the other side. This tight stacking against the pyridine ring probably contributes to the hyperthermostability of ArATPh. Compared with other ArATs, ArATPh has a novel substrate specificity, the order of preference being Tyr > Phe > Glu > Trp > His>> Met > Leu > Asp > Asn. Its relatively weak activity against Asp is due to lack of an arginine residue corresponding to Arg(292)* (where the asterisk indicates that this is a residues supplied by the other subunit of the dimer) in pig cytosolic aspartate aminotransferase. The enzyme recognizes the aromatic substrate by hydrophobic interaction with aromatic rings (Phe(121) and Tyr(59)*) and probably recognizes acidic substrates by a hydrophilic interaction involving a hydrogen bond network with Thr(264)*.

Cyclooctadienemethylplatinum complexes: synthesis, reactivity, molecular structure and spectroscopic properties of the organometallic hydroxoplatinum(II) complex [(COD)PtMe(OH)

Organometallic complexes of the type [(COD)PtMeL] (COD=1,5-cyclooctadiene, L=Cl, I, OH, Me, CH2C(O)CH3, CCPh, pyridine, N-methylpyrazinium) and the dinuclear complex [{(COD)PtMe}2(μ-CCCC)] were prepared and characterised by 1H-NMR spectroscopy. The crystal structures for the complexes with L=Cl, OH and Me are reported. The complex [(COD)PtMe(OH)] is a unique example of a platinum complex containing an olefin and a hydroxy ligand on the same metal centre. It reacts with CH acidic substrates to form PtC bonds. Representative reactions with acetone, phenylacetylene and 1,3-butadiyne and the corresponding products are reported.

Sphingomonas herbicidovorans MH: a versatile phenoxyalkanoic acid herbicide degrader.

Sphingomonas herbicidovorans MH was isolated from a dichlorprop-degrading soil column. It is able to grow on phenoxyalkanoic acid herbicides, such as mecoprop, dichlorprop, 2,4-D, MCPA, and 2,4-DB. Strain MH utilizes both enantiomers of the chiral herbicides mecoprop and dichlorprop as sole carbon and energy sources. Enantiomer-specific uptake systems are responsible for transporting the acidic substrates across the cell membrane. Catabolism is initiated by two enantiomer-specific alpha-ketoglutarate-dependent dioxygenases that catalyze the cleavage of the ether bond of the respective enantiomer to yield the corresponding phenol and pyruvate. Therefore selective degradation of the enantiomers of mecoprop and dichlorprop by strain MH is not only due to enantioselective catabolism but also to enantioselective transport.

The Yck2 yeast casein kinase 1 isoform shows cell cycle-specific localization to sites of polarized growth and is required for proper septin organization.

Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates. In yeast, the products of the redundant YCK1 and YCK2 genes are together essential for cell viability. Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis. Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle. Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis. At cytokinesis, GFP-Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes. The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant. The sites of GFP-Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization.

Examination of particulate macroporous hydrogels in an extracorporeal rat haemoperfusion model

A series of macroporous hydrogels has been synthesized, selected from a range of such materials in which the presence of functional groups has been shown to produce sorbent properties with respect to molecules having clinical significance in the field of liver support. The use of freeze-thaw polymerization, logether with inverse suspension polymerization in hexane, or in brine. enables macroporous beads ranging in size from 150 to 2000 μm. to be prepared from functional monomers exhibiting a range of chemical functionalities and aqueous solubilities. In order to investigate the behaviour of these rigid porous hydrophilic substrates in haemoperfusion. a rat model was used to explore various aspects of whole blood response. The materials were incorporated into an extracorporeal circuit linking the right cartoid artery and left jugular vein of male Sprague-Dawlcy rats. Erythrocyte, leucocyte and platelet levels were monitored over a 240 min haemoperfusion period. The most significant observation is that, apart from the strongly acidic polyacrylic acid substrate. matrix chemistry has relatively little effect on leucocyte or platelet response. The most important factors appear to be surface area, pore size and surface rugosity, which do produce measurable, but not dramatic. differences. This is encouraging for future work, since these variables may be manipulated by polymerization conditions.

Nickel(II)-induced activation of a carbon–nitrogen single bond(s) of a co-ordinated ligand by hydroxylated substrates: generation of mono- and bi-nuclear complexes with modified ligand frameworks †

In the presence of some hydroxylated compounds as substrates, complexations of nickel(II) with the pentadentate N4S ligand methyl 2-{2-bis[(3,5-dimethylpyrazol-1-ylmethyl)amino]propylamino}cyclopent-1-ene-1-carbodithioate (Hmmpcd) having a pair of flexible pyrazolyl arms proceeded through unusual reaction paths involving C–N bond cleavage process. For example, the product 1 obtained in methanol as substrate is a square planar compound with a modified ligand structure that includes a methoxy group provided by the solvent. With an acidic substrate viz. phenol the product is also a square planar compound 2 with one of the pendant arms of Hmmpcd being replaced by a hydrogen atom. On the other hand, in the presence of a basic substrate viz. ethanolamine the ligand undergoes more extensive breakdown to generate a binuclear complex 3 with a bridging pyrazolyl group. These complexes 1–3 were characterised by X-ray crystallography and 1H NMR spectroscopy. Compound 1 displayed a reversible one-electron NiII → NiI reduction at E½ ≈ –1.0 V vs. Ag–AgCl reference as established by cyclic voltammetry and combined coulometry/EPR experiments. Such reversibility of electron transfer is unusual for a square-planar nickel(II) compound in an acyclic ligand environment. Stabilisation of the reduced nickel(I) species by methoxy group co-ordination provides a probable explanation for this reversible electron transfer.

Optimization of l-lactic acid feeding for the production of poly- d-3-hydroxybutyric acid by Alcaligenes eutrophus in fed-batch culture

We investigated optimization of the feeding of l-lactic acid for the production of poly- d-3-hydroxybutyric acid [P(3HB)] by Alcaligenes eutrophus in a fed-batch culture system. An acidic substrate solution was fed automatically so as to maintain the pH of the culture liquid at 7.0. Feeding of a substrate solution containing 45% ( w v ) l-lactic acid, 6.2% ( w v ) sodium l-lactate, 5.8% ( w v ) ammonia water and 1.8% ( w v ) potassium phosphate [at a molar ratio of carbon to nitrogen ( C N molar ratio) of 10], allowed the l-lactate concentration in the culture liquid to be maintained at approximately 2 g/ l and the cell concentration reached 27.4 g/ l after 15 h of cultivation. To promote P(3HB) production, a two-stage fed-batch culture consisting of a culture for cell growth and one for P(3HB) accumulation was carried out. When the substrate solution, whose C N molar ratio was 23, was fed during the P(3HB) accumulation phase, the cell concentration and the P(3HB) content in the cells reached 103 g/ l and 57.6% ( w w ), respectively, in 51.5 h.

Protein kinase activities in ripening mango (Mangifera indica ) fruit tissue. II. Purification and characterization of a casein kinase I

A protein kinase (PK-II), phosphorylating casein, was purified from ripening mango, Mangifera indica L., fruit tissue. The purification procedure consisted of ammonium sulphate fractionation and sequential anion exchange-, dye-ligand, and gel filtration chromatography. The enzyme was purified over 500-fold to near homogeneity with a recovery of 4%. The purified enzyme had a specific activity of ca 1 µmol mg−1 protein min−1 with ATP as phosphoryl donor. SDS-PAGE results indicated a monomeric enzyme with molecular mass of 35 kDa. The protein kinase phosphorylated the acidic substrates casein and phosvitin, but had a very low activity with histones and protamine sulphate. The optimum pH and temperature for catalysis were determined to be 9.6 and 35°C, respectively. Mn2+ could not substitute for the Mg2+ needed for activity and Ca2+ had a slight stimulatory effect. Phospholipids, cAMP, calmodulin and the calmodulin inhibitor, calmidazolium, did not have any significant effect on activity, but the enzyme was inhibited by heparin and the specific inhibitor, CKI-7, (N-[2-aminoethyl]-5-chloroisoquinoline-8-sulphonamide). Autoradiographic studies revealed the ability of the protein kinase to autophosphorylate as well as the presence of endogenous protein substrates in the crude extract. Initial velocity studies with casein as substrate and product inhibition studies with ADP indicated a Km (ATP) and Km (casein) of 14 µ M and 0.18 mg ml−1, respectively, with a Ki (ADP) of 3.2 µM. The enzyme can be classified as a casein kinase I type of protein kinase (EC 2.7.10).

The Novel Substrate Recognition Mechanism Utilized by Aspartate Aminotransferase of the Extreme Thermophile Thermus thermophilus HB8

Aspartate aminotransferase (AspAT) is a unique enzyme that can react with two types of substrate with quite different properties, acidic substrates, such as aspartate and glutamate, and neutral substrates, although the catalytic group Lys-258 acts on both types of substrate. The dynamic properties of the substrate-binding site are indispensable to the interaction with hydrophobic substrates (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S. (1997) J. Biochem. (Tokyo) 122, 55-63). AspATs from various organisms are classified into two subgroups, Ia and Ib. The former includes AspATs from Escherichia coli and higher eukaryotes, whereas the latter includes those from Thermus thermophilus and many prokaryotes. The AspATs belonging to subgroup Ia each have an Arg-292 residue, which interacts with the distal carboxyl groups of dicarboxylic (acidic) substrates, but the functionally similar residue of subgroup Ib AspATs has not been identified. In view of the x-ray crystallographic structure of T. thermophilus AspAT, we expected Lys-109 to be this residue in the subgroup Ib AspATs and constructed K109V and K109S mutants. Replacing Lys-109 with Val or Ser resulted in loss of activity toward acidic substrates but increased that toward the neutral substrate, alanine, considerably. These results indicate that Lys-109 is a major determinant of the acidic substrate specificity of subgroup Ib AspATs. Kinetic analysis of the interactions with neutral substrates indicated that T. thermophilus AspAT is subject to less steric hindrance and its substrate-binding pocket has a more flexible conformation than E. coli AspAT. A flexible active site in the rigid T. thermophilus AspAT molecule may explain its high activity even at room temperature.

Crystal structures of Paracoccus denitrificans aromatic amino acid aminotransferase: a substrate recognition site constructed by rearrangement of hydrogen bond network

Aminotransferase reversibly catalyzes the transamination reaction by a ping-pong bi-bi mechanism with pyridoxal 5′-phosphate (PLP) as a cofactor. Various kinds of aminotransferases developing into catalysts for particular substrates have been reported. Among the aminotransferases, aromatic amino acid aminotransferase (EC 2.6.1.57) catalyzes the transamination reaction with both acidic substrates and aromatic substrates. To elucidate the multiple substrate recognition mechanism, we determined the crystal structures of aromatic amino acid aminotransferase from Paracoccus denitrificans (pdAroAT): unliganded pdAroAT, pdAroAT in a complex with maleate as an acidic substrate analog, and pdAroAT in a complex with 3-phenylpropionate as an aromatic substrate analog at 2.33 Å, 2.50 Å and 2.30 Å resolution, respectively.The pdAroAT molecule is a homo-dimer. Each subunit has 394 amino acids and one PLP and is divided into small and large domains. The overall structure of pdAroAT is essentially identical to that of aspartate aminotransferase (AspAT) which catalyzes the transamination reaction with only an acidic amino acid. On binding the acidic substrate analog, arginine 292 and 386 form end-on salt bridges with carboxylates of the analog. Furthermore, binding of the substrate induces the domain movement to close the active site. The recognition mechanism for the acidic substrate analog in pdAroAT is identical to that observed in AspAT. Binding of the aromatic substrate analog causes reorientation of the side-chain of the residues, lysine 16, asparagine 142, arginine 292∗ and serine 296∗, and changes in the position of water molecules in the active site to form a new hydrogen bond network in contrast to the active site structure of pdAroAT in the complex with an acidic substrate analog. Consequently, the rearrangement of the hydrogen bond network can form recognition sites for both acidic and aromatic side-chains of the substrate without a conformational change in the backbone structure in pdAroAT.

Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases

Aromatic amino acid aminotransferase (AroAT) and aspartate aminotransferase (AspAT) are known as dual-substrate enzymes, which can bind acidic and hydrophobic substrates in the same pocket (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S. (1997) J. Biochem. (Tokyo) 122, 55-63). In order to elucidate the mechanism of hydrophobic substrate recognition, kinetic and thermodynamic analyses using substrates with different hydrophobicities were performed. They revealed that 1) amino acid substrate specificity (kmax/Kd) depended on the affinity for the substrate (1/Kd) and 2) binding of the hydrophobic side chain was enthalpy-driven, suggesting that van der Waals interactions between the substrate-binding pocket and hydrophobic substrate predominated. Three-dimensional structures of AspAT and AroAT bound to alpha-aminoheptanoic acid were built using the homology modeling method. A molecular dynamic simulation study suggested that the outward-facing position of the Arg292 side chain was the preferred state to a greater extent in AroAT than AspAT, which would make the hydrophobic substrate bound state of the former more stable. Furthermore, AroAT appeared to have a more flexible conformation than AspAT. Such flexibility would be expected to reduce the energetic cost of conformational rearrangement induced by substrate binding. These two mechanisms (positional preference of Arg and flexible conformation) may account for the high activity of AroAT toward hydrophobic substrates.

Separation of acidic protein tyrosine kinase substrates by strong anion-exchange chromatography

A series of heptapeptide sequences containing two glutamic and one aspartic acid residues, synthesized as substrates or inhibitors of different tyrosine kinases, and their phosphorylated or phosphonate analogs, were characterized analytically by strong anion-exchange high-performance liquid chromatography. Peptides of slightly different acidity can be separated under the experimental conditions used and the elution order increased in parallel with the number of acidic functions present in the sequence. The eluents and the characteristics of strong anion-exchange columns permit the scale-up of the method described to preparative purification.

Uranium amides as precursors to cationic and/or pentavalent compounds

The uranium chloroamide complexes [U(NEt 2) n Cl 4− n ] ( n=1, 2) were isolated from the comproportionation reactions of [U(NEt 2) 4] and UCl 4. The protonolysis reaction of the M–NR 2 bond (M=Ti, Zr, Hf, U; R=Me, Et, SiMe 3) with the ammonium salt NHR 3′BPh 4 (R′=Et, Me) constitutes an efficient and practical route to cationic complexes. Thus were prepared a series of organometallic uranium cations in the +3, +4 and +5 oxidation states. The reactions of these cationic compounds with nucleophiles, proton acidic substrates and unsaturated molecules have been developed. The dialkylamide ligand was useful to stabilize unique examples of neutral and cationic complexes of uranium in the +5 oxidation state.

Palladium catalysed reactions of allenes, carbon monoxide and nucleophiles

Hydridopalladium(II) species generated in situ by oxidative addition of Pd(0) to acetic acid or acidic hydroxyl substrates (phenols, oximes) catalyse the termolecular assembly of allenes, CO and amines (primary, secondary) or oxygen nucleophiles to give methacrylamides or methacrylate esters and derivatives thereof in good to excellent yield.