Interfacial thermodynamics of protein adsorption, ion co-adsorption and ion binding in solution. II. Model interpretation of ion exchange in lysozyme chromatography

In this paper we present a model for the ion exchange effects in protein adsorption. The model is applied to chromatography of lysozyme on strong cation exchanger ‘mono S’. The experimental and general thermodynamic aspects have been discussed in Part 1, the preceding paper. The main modelling assumptions are (i) the charge regulation is confined to the small layer of contact between adsorbed protein and exchanger surface, (ii) the contact layer as a whole is electroneutral and (iii) the number of protein acid/base groups and exchanger surface acid groups which participate in the ion exchange is proportional to the area of the contact layer. The model is fitted to the experimental data by adjustment of only two or three parameters. The experimental co-adsorption numbers are very well reproduced. A few conspicuous features emerge: (i) the number of protein acid/base groups and exchanger surface acid groups in the contact layer varies with the medium conditions, such that the number is higher when the interaction between protein and exchanger surface is stronger. (ii) There is indirect evidence for structural alterations in the upper layers of the exchanger surface: the adsorbed protein is probably partly ‘buried’ in the surface.     

Changing the substrate specificity of a chitooligosaccharide oxidase from Fusarium graminearum by model-inspired site-directed mutagenesis

Chitooligosaccharide oxidase (ChitO) catalyzes the oxidation of C1 hydroxyl moieties on chitooligosaccharides and in this way displays a different substrate preference as compared to other known oligosaccharide oxidases. ChitO was identified in the genome of Fusarium graminearum and a structural model revealed that one active site residue (Q268) was likely to be involved in the recognition of the N-acetyl moiety on the chitooligosaccharide substrates. The substrate specificity of wild type ChitO and the Q268R mutant were examined and confirmed that Q268 is indeed involved in N-acetyl recognition.     

Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic halohydrins. To understand the kinetic mechanism and enantioselectivity of the enzyme, steady-state and pre-steady-state kinetic analysis was performed with p-nitro-2-bromo-1-phenylethanol (PNSHH) as a model substrate. Steady-state kinetic analyses indicated that the k(cat) of the enzyme with the (R)-enantiomer (22 s(-1)) is 3-fold higher than with the (S)-enantiomer and that the K(m) for the (R)-enantiomer (0.009 mM) is about 45-fold lower than that for the (S)-enantiomer, resulting in a high enantiopreference for the (R)-enantiomer. Product inhibition studies revealed that HheC follows an ordered Uni Bi mechanism for both enantiomers, with halide as the first product to be released. To identify the rate-limiting step in the catalytic cycle, pre-steady-state experiments were performed using stopped-flow and rapid-quench methods. The results revealed the existence of a pre-steady-state burst phase during conversion of (R)-PNSHH, whereas no such burst was observed with the (S)-enantiomer. This indicates that a product release step is rate-limiting for the (R)-enantiomer but not for the (S)-enantiomer. This was further examined by doing single-turnover experiments, which revealed that during conversion of the (R)-enantiomer the rate of bromide release is 21 s(-1). Furthermore, multiple turnover analyses showed that the binding of (R)-PNSHH is a rapid equilibrium step and that the rate of formation of product ternary complex is 380 s(-1). Taken together, these findings enabled the formulation of an ordered Uni Bi kinetic mechanism for the conversion of (R)-PNSHH by HheC in which all of the rate constants are obtained. The high enantiopreference for the (R)-enantiomer can be explained by weak substrate binding of the (S)-enantiomer and a lower rate of reaction at the active site.     

Structural analysis of flavinylation in vanillyl-alcohol oxidase

Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8alpha-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K(d) = 1.8 and 2.1 microm, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-)(1), K(m) = 40 microm) than the wild-type enzyme. The crystal structures of both the holo and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-61 plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site.     

Discovery of a novel styrene monooxygenase originating from the metagenome

Oxygenases form an interesting class of biocatalysts, as they typically perform oxygenations with exquisite chemo-, regio-, and/or enantioselectivity. It has been observed that, once heterologously expressed in Escherichia coli, some oxygenases are able to form the blue pigment indigo. We have exploited this characteristic to screen a metagenomic library derived from loam soil and identified a novel oxygenase. This oxygenase shows 50% sequence identity with styrene monooxygenases from pseudomonads (StyA). Only a limited number of homologs can be found in the genome sequence database, indicating that this biocatalyst is a member of a relatively small family of bacterial monooxygenases. The newly identified monooxygenase catalyzes the epoxidation of styrene and styrene derivatives and forms the corresponding (S)-epoxides with excellent enantiomeric excess [e.g., (S)-styrene oxide is formed with >99% enantiomeric excess, ee] and therefore is named styrene monooxgenase subunit A (SmoA). SmoA shows high enantioselectivity towards aromatic sulfides [e.g., (R)-ethyl phenyl sulfoxide is formed with 92% ee]. This excellent enantioselectivity in combination with the moderate sequence identity forms a clear indication that SmoA from a metagenomic origin represents a new enzyme within the small family of styrene monooxygenases.     

Interactions among oscillatory pathways in NF-kappa B signaling

Background  

Sustained stimulation with tumour necrosis factor alpha (TNF-alpha) induces substantial oscillations—observed at both the single cell and population levels—in the nuclear factor kappa B (NF-kappa B) system. Although the mechanism has not yet been elucidated fully, a core system has been identified consisting of a negative feedback loop involving NF-kappa B (RelA:p50 hetero-dimer) and its inhibitor I-kappa B-alpha. Many authors have suggested that this core oscillator should couple to other oscillatory pathways.     

Biocatalytic properties of Baeyer–Villiger monooxygenases in aqueous–organic media

The biocatalytic properties of three Baeyer–Villiger monooxygenases (phenylacetone monooxygenase, 4-hydroxyacetophenone monooxygenase and ethionamide monooxygenase) in a variety of aqueous–organic media were studied using organic sulfides as substrates. The influence of the nature and the concentration of the solvents, as well as of the substrates, on the activity and enantioselectivity of the enzymes was investigated in detail. Solvents were found to decrease, to a different extent, enzyme activity. High increases of enantioselectivity and also reversal of enantiopreference were observed depending on the enzyme and on the nature of the solvent and the substrate employed.     

Animal behavior frozen in time: gregarious behavior of Early Jurassic lobsters within an ammonoid body chamber

Direct animal behavior can be inferred from the fossil record only in exceptional circumstances. The exceptional mode of preservation of ammonoid shells in the Posidonia Shale (Lower Jurassic, lower Toarcian) of Dotternhausen in southern Germany, with only the organic periostracum preserved, provides an excellent opportunity to observe the contents of the ammonoid body chamber because this periostracum is translucent. Here, we report upon three delicate lobsters preserved within a compressed ammonoid specimen of Harpoceras falciferum. We attempt to explain this gregarious behavior. The three lobsters were studied using standard microscopy under low angle light. The lobsters belong to the extinct family of the Eryonidae; further identification was not possible. The organic material of the three small lobsters is preserved more than halfway into the ammonoid body chamber. The lobsters are closely spaced and are positioned with their tails oriented toward each other. The specimens are interpreted to represent corpses rather than molts. The lobsters probably sought shelter in preparation for molting or against predators such as fish that were present in Dotternhausen. Alternatively, the soft tissue of the ammonoid may have been a source of food that attracted the lobsters, or it may have served as a long-term residency for the lobsters (inquilinism). The lobsters represent the oldest known example of gregariousness amongst lobsters and decapods in the fossil record. Gregarious behavior in lobsters, also known for extant lobsters, thus developed earlier in earth's history than previously known. Moreover, this is one of the oldest known examples of decapod crustaceans preserved within cephalopod shells.     

Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat

Chemical control of Septoria leaf blotch, caused by Mycosphaerella graminicola, is essential to ensure wheat yield and food security in most European countries. Mycosphaerella graminicola has developed resistance to several classes of fungicide and, with the efficacy of azoles gradually declining over time, new modes of action and/or improvements in host varietal resistance are urgently needed to ensure future sustainable disease control. Several new‐generation carboxamide fungicides with broad‐spectrum activity have recently been introduced into the cereal market. Carboxamides inhibit succinate dehydrogenase (Sdh) of the mitochondrial respiratory chain (complex II) but, because of their single‐site specificity, these fungicides may be prone to resistance development. The objective of this study was to assess the risk of resistance development to different Sdh inhibitor (SDHI) fungicides in M. graminicola. UV mutagenesis was conducted to obtain a library of carboxin‐resistant mutants. A range of SDHI resistance‐conferring mutations was found in Sdh subunits B, C and D. Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations. Most of the amino acid residues identified (e.g. B‐S221P/T, B‐H267F/L/N/Y, B‐I269V and D‐D129E/G/T) are directly involved in forming the cavity in which SDHI fungicides bind. Docking studies of SDHI fungicides in structural models of wild‐type and mutated Sdh complexes also indicated which residues were important for the binding of different SDHI fungicides and showed a different binding for fluopyram. The predictive power of the model was also shown. Further diagnostic development, enabling the detection of resistant alleles at low frequencies, and cross‐resistance studies will aid the implementation of anti‐resistance strategies to prolong the cost‐effectiveness and lifetime of SDHI fungicides.     

The enigmatic reaction of flavins with oxygen

The reaction of flavoenzymes with oxygen remains a fascinating area of research because of its relevance for reactive oxygen species (ROS) generation. Several exciting recent studies provide consistent mechanistic clues about the specific functional and structural properties of the oxidase and monooxygenase flavoenzymatic systems. Specifically, the spatial arrangement of the reacting oxygen that is in direct contact with the flavin group is emerging as a crucial factor that differentiates between oxidase and monooxygenase enzymes. A challenge for the future will be to use these emerging concepts to rationally engineer flavoenzymes, paving the way to new research avenues with far-reaching implications for oxidative biocatalysis and metabolic engineering.