Enzymatic peptide synthesis by new supported biocatalysts

Summary It is well known that proteases can be successfully used for peptide synthesis in aqueous medium or in water-containing organic solvents. Copolymerized of acrylated derivatives of-chymotrypsin and polyethylene glycol (PEG) have been prepared and used as biocatalysts for the synthesis of model peptides in organic solvents containing a very low quantity of water. Trypsin has been similarly treated and used as a new biocatalyst particularly for the coupling of non-specific substrates.     

Applications of mass spectrometry in screening for new biocatalysts

Each biocatalyst screen is unique, defined by the combination of factors involved in the screen, including the number and type of biocatalysts in the screening collection, substrate chemistry and the type of assay. Advances in the technology surrounding mass spectrometry — in software, in ionization sources and interfaces and in engineering, which allows smaller mass spectrometry systems and narrow bore HPLC — have made the application of this versatile technology in screening assays possible. A mass spectrometric assay provides sensitive, specific, quantitative, high-throughput detection of new biocatalyst activities. Examples of these applications are presented and potential pitfalls are discussed.     

Characterisation of the ATP-dependent phosphofructokinase gene family from Arabidopsis thaliana

Plants possess two different types of phosphofructokinases, an ATP-dependent (PFK) and a pyrophosphate-dependent form (PFP). While plant PFPs have been investigated in detail, cDNA clones coding for PFK have not been identified in Arabidopsis thaliana. Searching the A. thaliana genome revealed 11 putative members of a phosphofructokinase gene family. Among those, four sequences showed high homology to the alpha- or beta-subunits of plant PFPs. Seven cDNAs resulted in elevated PFK, but not PFP activity after transient expression in tobacco leaves suggesting that they encode Arabidopsis PFKs. RT-PCR revealed different tissue-specific expression of the individual forms. Furthermore, analysis of GFP fusion proteins indicated their presence in different sub-cellular compartments.     

A new family of bacterial condensins

Condensins play a central role in global chromatin organization. In bacteria, two families of condensins have been identified, the MukBEF and SMC-ScpAB complexes. Only one of the two complexes is usually found in a given species, giving rise to a paradigm that a single condensin organizes bacterial chromosomes. Using sequence analysis, we identified a third family of condensins, MksBEF (MukBEF-like SMC proteins), which is broadly present in diverse bacteria. The proteins appear distantly related to MukBEF, have a similar operon organization and similar predicted secondary structures albeit with notably shorter coiled-coils. All three subunits of MksBEF exhibit significant sequence variation and can be divided into a series of overlapping subfamilies. MksBEF often coexists with the SMC-ScpAB, MukBEF and, sometimes, other MksBEFs. In Pseudomonas aeruginosa, both SMC and MksB contribute to faithful chromosome partitioning, with their inactivation leading to increased frequencies of anucleate cells. Moreover, MksBEF can complement anucleate cell formation in SMC-deficient cells. Purified PaMksB showed activities typical for condensins including ATP-modulated DNA binding and condensation. Notably, DNA binding by MksB is negatively regulated by ATP, which sets it apart from other known SMC proteins. Thus, several specialized condensins might be involved in organization of bacterial chromosomes.     

Stability of biocatalysts

Despite their many favorable qualities, the marginal stability of biocatalysts in many types of reaction media often has prevented or delayed their implementation for industrial-scale synthesis of fine chemicals and pharmaceuticals. Consequently, there is great interest in understanding effects of solution conditions on protein stability, as well as in developing strategies to improve protein stability in desired reaction media. Recent methods include novel chemical modifications of protein, lyophilization in the presence of additives, and physical immobilization on novel supports. Rational and combinatorial protein engineering techniques have been used to yield unmodified proteins with exceptionally improved stability. Both have been aided by the development of computational tools and structure-guided heuristics aimed at reducing library sizes that must be generated and screened to identify improved mutants. The number of parameters used to indicate protein stability can complicate discussions and investigations, and care should be taken to identify whether thermodynamic or kinetic stability limits the observed stability of proteins. Although the useful lifetime of a biocatalyst is dictated by its kinetic stability, only 6% of protein stability studies use kinetic stability measures. Clearly, more effort is needed to study how solution conditions impact protein kinetic stability.     

A new family of carbon-nitrogen hydrolases.

Using computer methods for database search and multiple alignment, statistically significant sequence similarities were identified between several nitrilases with distinct substrate specificity, cyanide hydratases, aliphatic amidases, beta-alanine synthase, and a few other proteins with unknown molecular function. All these proteins appear to be involved in the reduction of organic nitrogen compounds and ammonia production. Sequence conservation over the entire length, as well as the similarity in the reactions catalyzed by the known enzymes in this family, points to a common catalytic mechanism. The new family of enzymes is characterized by several conserved motifs, one of which contains an invariant cysteine that is part of the catalytic site in nitrilases. Another highly conserved motif includes an invariant glutamic acid that might also be involved in catalysis.     

A new family of 2-hydroxyacid dehydrogenases

The NADH-dependent hydroxypyruvate reductase from cucumber and the pdxB gene product of E. coli display significant homology to E. coli D-3-phosphoglycerate dehydrogenase. In contrast, these proteins do not display much similarity with other oxidoreductases or with other 2-hydroxyacid dehydrogenases in particular. On the basis of their relatedness and the structure of their substrates, these three enzymes constitute a new family of 2-hydroxyacid dehydrogenases distinct from malate and lactate dehydrogenase.     

A new family of insect tyramine receptors

The Drosophila Genome Project database contains a gene, CG7431, annotated to be an "unclassifiable biogenic amine receptor." We have cloned this gene and expressed it in Chinese hamster ovary cells. After testing various ligands for G protein-coupled receptors, we found that the receptor was specifically activated by tyramine (EC(50), 5x10(-7)M) and that it showed no cross-reactivity with beta-phenylethylamine, octopamine, dopa, dopamine, adrenaline, noradrenaline, tryptamine, serotonin, histamine, and a library of 20 Drosophila neuropeptides (all tested in concentrations up to 10(-5) or 10(-4)M). The receptor was also expressed in Xenopus oocytes, where it was, again, specifically activated by tyramine with an EC(50) of 3x10(-7)M. Northern blots showed that the receptor is already expressed in 8-hour-old embryos and that it continues to be expressed in all subsequent developmental stages. Adult flies express the receptor both in the head and body (thorax/abdomen) parts. In addition to the Drosophila tyramine receptor gene, CG7431, we found another closely related Drosophila gene, CG16766, that probably also codes for a tyramine receptor. Furthermore, we annotated similar tyramine-like receptor genes in the genomic databases from the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera. These four tyramine or tyramine-like receptors constitute a new receptor family that is phylogenetically distinct from the previously identified insect octopamine/tyramine receptors. The Drosophila tyramine receptor is, to our knowledge, the first cloned insect G protein-coupled receptor that appears to be fully specific for tyramine.     

A new taxonomy of the family Teloschistaceae

The lichen family Teloschistaceae is one of the larger families of lichenized fungi. Currently it includes one very large heterogenous genus, Caloplaca, with some 1000 or more species with a vast variation in morphology, anatomy and chemistry. The rest of the family is split into 10–15 smaller genera, each with 20 or fewer species. There is no modern classification of the family based on molecular data. Here we attempt a first phylogenetic evaluation of a large part of the family, including a total of 337 species. Of these, 162 were used in a combined phylogenetic analysis of the ribosomal RNA sequence markers nrITS, nrLSU and mrSSU, using Bayesian inference. We also analysed all species using nrITS data, split into four different analyses. As a result we propose a new classification of the family, where a total of 39 genera are recognized, of which 31 are newly described or resurrected. The new genera are: Athallia, Austroplaca, Bryoplaca, Calogaya, Cerothallia, Flavoplaca, Gondwania, Haloplaca, Orientophila, Pachypeltis, Parvoplaca, Rufoplaca, Shackletonia, Scutaria, Sirenophila, Solitaria, Squamulea, Stellarangia, Teloschistopsis, Usnochroma, Variospora, Villophora and Wetmoreana. Resurrected genera are Blastenia, Dufourea, Follmannia, Gyalolechia, Leproplaca, Polycauliona, Pyrenodesmia and Xanthocarpia. The species Orientophila subscopularis is described as new. A third subfamily, Teloschistoideae, is proposed to accommodate the genus Teloschistes and related genera, parallel to the two previously recognized subfamilies Xanthorioideae and Caloplacoideae. We also show the large plasticity in both morphological and anatomical characters between closely related species within genera, indicating the low value of these as evolutionary markers. The secondary chemistry is a better marker in some parts of the family. We recognize a large number of geographically delimited clades with clear centres of evolution, but often showing large variation in morphology and anatomy.     

Glycosyltransferases as biocatalysts

Glycosyltransferases are useful synthetic tools for the preparation of natural oligosaccharides, glycoconjugates and their analogues. High expression levels of recombinant enzymes have allowed their use in multi-step reactions, on mg to multi-gram scales. Since glycosyltransferases are tolerant with respect to utilizing modified donors and acceptor substrates they can be used to prepare oligosaccharide analogues and for diversification of natural products. New sources of enzymes are continually discovered as genomes are sequenced and they are annotated in the Carbohydrate Active Enzyme (CAZy) glycosyltransferase database. Glycosyltransferase mutagenesis, domain swapping and metabolic pathway engineering to change reaction specificity and product diversification are increasingly successful due to advances in structure-function studies and high throughput screening methods.     

Functional delineation of three groups of the ATP-dependent family of chromatin remodeling enzymes

ATP-dependent chromatin remodeling enzymes antagonize the inhibitory effects of chromatin. We compare six different remodeling complexes: ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, and dNURF. We find that each complex uses similar amounts of ATP to remodel nucleosomal arrays at nearly identical rates. We also perform assays with arrays reconstituted with hyperacetylated or trypsinized histones and isolated histone (H3/H4)(2) tetramers. The results define three groups of the ATP-dependent family of remodeling enzymes. In addition we investigate the ability of an acidic activator to recruit remodeling complexes to nucleosomal arrays. We propose that ATP-dependent chromatin remodeling enzymes share a common reaction mechanism and that a key distinction between complexes is in their mode of regulation or recruitment.     

A new murine Ig VH gene family

A novel murine VH gene family, termed VH10, has been found and characterized. Based on RFLP analysis, this family exhibits extensive polymorphism among inbred strains of mice and encompasses two to five members, depending on the Igh haplotype. Analyses of recombinant inbred strains suggest a map position of this family 5' to the 7183 and Q52 VH gene families. A VH10 gene has been found to encode anti-DNA autoantibodies from lupus mice; another one may be a pseudogene.