Laboratory evolution of laccase for substrate specificity

A laccase, CotA, from Bacillus subtilis was engineered using a combination of rational and directed evolution approaches. CotA is a generalist, an enzyme with broad specificity, and it was optimized to be a specialist, an enzyme with narrowed specificity. Wild-type CotA oxidizes ABTS (ABTS = diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and SGZ (SGZ = 4-hydroxy-3,5-dimethoxy-benzaldehyde azine), and it was engineered for increased specificity for ABTS. Based on the ABTS-bound crystal structure of CotA, 19 amino acids are within 6 Å of ABTS, and they were simultaneously randomized. A mutant was identified that was 132 times more specific for ABTS. Unexpectedly, the variant was found to acquire enhanced thermal stability. The half-life for the heat inactivation (t_1/2) at 80 °C was increased by 62 min for the mutant. Laccases have several applications in biotechnology, which include pulp bleaching, biosensors, bioremediation, and biofuel cells. The substrate specificity of CotA is moldable and the enzyme can be tailored to oxidize a variety of target molecules for specific purposes.     

Genome plasticity during seed germination in Festuca arundinacea

 Feulgen/DNA cytophotometric determinations were carried out on early prophases in the meristems of seedlings obtained by germinating seeds of different accessions of Festuca arundinacea at 10°C, 20°C, or 30°C. Feulgen/DNA contents increased significantly with the increase in the temperature of seed germination. In each accession, the greater the increase in absorption in seedlings obtained at 30°C, the lower the absorption in seedlings obtained at 10°C. In contrast, Feulgen/DNA contents did not undergo changes when the temperature was altered at developmental stages other than seed germination. The results of molecular hybridizations (slot blots) indicated that the redundancy of repeated DNA sequences belonging to two families having C_ot ranges of 0–2×10^-1 and 2×10^-1 −2×10⁰, respectively, was significantly higher in the genome of seedlings obtained at 30°C than in that of seedlings obtained at 10°C. When centrifuged to equilibrium in CsCl density gradients, the DNA extracted from seedlings obtained at 30°C formed a heavier and a lighter shoulder with buoyant densities of 1.707 g/ml and 1.692 g/ml, respectively, in addition to the main band (1.701 g/ml). Only a less apparent shoulder banding at 1.706 g/ml was formed by the DNA extracted from seedlings obtained at 10°C. After seed germination in the presence of [³H]-thymidine for 24 h at 30°C, most of radioactivity was found in the guanine + cytosine- or adenine+thymine-enriched DNA fractions, which formed the two shoulders in the density profile. In contrast, only guanine+cytosine-enriched fractions, which formed the heavier shoulder, were preferentially labelled in the DNA from seedlings obtained at 10°C. These results prove that fluid domains do exist in the nuclear DNA of F. arundinacea. These DNA domains are capable of rapid, quantitative alterations, which represent the direct responses of the genome to developmental and environmental stimuli. Seed germination appears to be a limited, specific period in development within which the adaptive response to temperature variations can be put into effect. Received: 9 August 1996 / Accepted: 23 August 1996     

Proteomic analysis identified N-cadherin, clusterin, and HSP27 as mediators of SPARC (secreted protein, acidic and rich in cysteines) activity in melanoma cells

Secreted protein, acidic and rich in cysteines (SPARC) is a secreted protein associated with increased aggressiveness of different human cancer types. In order to identify downstream mediators of SPARC activity, we performed a 2-DE proteomic analysis of human melanoma cells following antisense-mediated downregulation of SPARC expression. We found 23/504 differential spots, 15 of which were identified by peptide fingerprinting analysis. Three of the differential proteins (N-cadherin (N-CAD), clusterin (CLU), and HSP27) were validated by immunoblotting, confirming decreased levels of N-CAD and CLU and increased amounts of HSP27 in conditioned media of cells with diminished SPARC expression. Furthermore, transient knock down of SPARC expression in melanoma cells following adenoviral-mediated transfer of antisense RNA confirmed these changes. We next developed two different RNAs against SPARC that were able to inhibit in vivo melanoma cell growth. Immunoblotting of the secreted fraction of RNAi-transfected melanoma cells confirmed that downregulation of SPARC expression promoted decreased levels of N-CAD and CLU and increased secretion of HSP27. Transient re-expression of SPARC in SPARC-downregulated cells reverted extracellular N-CAD, CLU, and HSP27 to levels similar to those in the control. These results constitute the first evidence that SPARC, N-CAD, CLU, and HSP27 converge in a unique molecular network in melanoma cells.     

Role of futC slipped strand mispairing in Helicobacter pylori Lewisy phase variation

The O antigen of the Helicobacter pylori lipopolysaccharide is composed of repeating units of fucosylated Lewis (Le) antigens. The alpha(1,2)-fucosyltransferase (futC) of H. pylori, which catalyzes the conversion of Le(x) to Le(y) by addition of fucose, is subject to slipped-strand mispairing involving a homonucleotide (poly-C) tract. To explore the distribution of Le phenotypes within H. pylori cells grown in vitro, 379 single colonies of strain J166 were examined for Le expression. Two major populations with reciprocal Le(x)/Le(y) phenotypes were identified. Phenotypes correlated with futC frame status, suggesting that strain J166 represents a mixed population with respect to futC poly-C tract length, which was confirmed by a translational reporter. After hundreds of generations in vitro, phenotypes did not change significantly, indicating that the observed J166 Le diversity reflects the founding population. Since slipped-strand mispairing in the futC poly-C tract was postulated to explain the Le(y) phenotypic change observed in J166 derivative strain 98-169 isolated 10 months after rhesus monkey challenge, in trans complementation with in-frame futC was performed. Le(y) synthesis was restored and Le(x) expression was reciprocally lowered. From these studies, we confirmed the principal role of futC slipped-strand mispairing in Le antigenic variation in vitro and in vivo.