Directed evolution of a (βα)8-barrel enzyme to catalyze related reactions in two different metabolic pathways

Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC 5.3.1.16) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC 5.3.1.24), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (βα)8-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (βα)8-barrel enzymes are very suitable for the design of new catalytic activities.

Photochemical protease: Site-specific photocleavage of hen egg lysozyme and bovine serum albumin

Site-specific photocleavage of hen egg lysozyme and bovine serum albumin (BSA) by N-(l-phenylalanine)-4-(1-pyrene)butyramide (Py-Phe) is reported. Py-Phe binds to lysozyme and BSA with binding constants 2.2 ± 0.3 × 105 M−1 and 6.5 ± 0.4 × 107 M−1, respectively. Photocleavage of lysozyme and BSA was achieved with high specificity when a mixture of protein, Py-Phe, and an electron acceptor, cobalt(III) hexammine (CoHA), was irradiated at 344 nm. Quantum yields of photocleavage of lysozyme and BSA were 0.26 and 0.0021, respectively. No protein cleavage was observed in the absence of Py-Phe, CoHA, or light. N-terminal sequencing of the protein fragments indicated a single cleavage site of lysozyme between Trp-108 and Val-109, whereas the cleavage of BSA was found to be between Leu-346 and Arg-347. Laser flash photolysis studies of a mixture of protein, Py-Phe, and CoHA showed a strong transient with absorption centered at ≈460 nm, corresponding to pyrene cation radical. Quenching of the singlet excited state of Py-Phe by CoHA followed by the reaction of the resulting pyrenyl cation radical with the protein backbone may be responsible for the protein cleavage. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.

Reactions of participants to the results of a randomised controlled trial: exploratory study

Objectives: To assess views of parents of babies who participated in a neonatal trial, about feedback of trial results.

BIND—The Biomolecular Interaction Network Database

The Biomolecular Interaction Network Database (BIND; http://binddb.org) is a database designed to store full descriptions of interactions, molecular complexes and pathways. Development of the BIND 2.0 data model has led to the incorporation of virtually all components of molecular mechanisms including interactions between any two molecules composed of proteins, nucleic acids and small molecules. Chemical reactions, photochemical activation and conformational changes can also be described. Everything from small molecule biochemistry to signal transduction is abstracted in such a way that graph theory methods may be applied for data mining. The database can be used to study networks of interactions, to map pathways across taxonomic branches and to generate information for kinetic simulations. BIND anticipates the coming large influx of interaction information from high-throughput proteomics efforts including detailed information about post-translational modifications from mass spectrometry. Version 2.0 of the BIND data model is discussed as well as implementation, content and the open nature of the BIND project. The BIND data specification is available as ASN.1 and XML DTD.

Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-β-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.

Comparison of the Ability of Partially N-Acetylated Chitosans and Chitooligosaccharides to Elicit Resistance Reactions in Wheat Leaves1

Chitin, a linear polysaccharide composed of (1→4)-linked 2-acetamido-2-deoxy-β-d-glucopyranose (GlcNAc) residues, and chitosan, the fully or partially N-acetylated, water-soluble derivative of chitin composed of (1→4)-linked GlcNAc and 2-amino-2-deoxy-β-d-glucopyranose (GlcN), have been proposed as elicitors of defense reactions in higher plants. We tested and compared the ability of purified (1→4)-linked oligomers of GlcNAc (tetramer to decamer) and of GlcN (pentamer and heptamer) and partially N-acetylated chitosans with degrees of acetylation (DA) of 1%, 15%, 35%, 49%, and 60% and average degrees of polymerization between 540 and 1100 to elicit phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin deposition, and microscopically and macroscopically visible necroses when injected into the intercellular spaces of healthy, nonwounded wheat (Triticum aestivum L.) leaves. Purified oligomers of (1→4)-linked GlcN were not active as elicitors, whereas purified oligomers of (1→4)-linked GlcNAc with a degree of polymerization ≥ 7 strongly elicited POD activities but not PAL activities. Partially N-acetylated, polymeric chitosans elicited both PAL and POD activities, and maximum elicitation was observed with chitosans of intermediate DAs. All chitosans but not the chitin oligomers induced the deposition of lignin, the appearance of necrotic cells exhibiting yellow autofluorescence under ultraviolet light, and macroscopically visible necroses; those with intermediate DAs were most active. These results suggest that different mechanisms are involved in the elicitation of POD activities by GlcNAc oligomers, and of PAL and POD activities by partially N-acetylated chitosan polymers and that both enzymes have to be activated for lignin biosynthesis and ensuing necrosis to occur.

Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat1

Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.

Photochemical energy conversion in a helical oligoproline assembly.

A general method is described for constructing a helical oligoproline assembly having a spatially ordered array of functional sites protruding from a proline-II helix. Three different redox-active carboxylic acids were coupled to the side chain of cis-4-amino-L-proline. These redox modules were incorporated through solid-phase peptide synthesis into a 13-residue helical oligoproline assembly bearing in linear array a phenothiazine electron donor, a tris(bipyridine)ruthenium(II) chromophore, and an anthraquinone electron acceptor. Upon transient 460-nm irradiation in acetonitrile, this peptide triad formed with 53% efficiency an excited state containing a phenothiazine radical cation and an anthraquinone radical anion. This light-induced redox-separated state had a lifetime of 175 ns and stored 1.65 eV of energy.

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.