Peptidoglycan N-acetylglucosamine deacetylases from Bacillus cereus, highly conserved proteins in Bacillus anthracis

The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain 10 polysaccharide deacetylase homologues. Six of these homologues have been proposed to be peptidoglycan N-acetylglucosamine deacetylases. Two of these genes, namely bc1960 and bc3618, have been cloned and expressed in Escherichia coli, and the recombinant enzymes have been purified to homogeneity and further characterized. Both enzymes were effective in deacetylating cell wall peptidoglycan from the Gram(+) Bacillus cereus and Bacillus subtilis and the Gram(-) Helicobacter pylori as well as soluble chitin substrates and N-acetylchitooligomers. However, the enzymes were not active on acetylated xylan. These results provide insight into the substrate specificity of carbohydrate esterase family 4 enzymes. It was revealed that both enzymes deacetylated only the GlcNAc residue of the synthetic muropeptide N-acetyl-D-glucosamine-(beta-1,4)-N-acetylmuramyl-L-alanine-D-isoglutamine. Analysis of the constituent muropeptides of peptidoglycan from B. subtilis and H. pylori resulting from incubation of the enzymes BC1960 and BC3618 with these polymers and subsequent hydrolysis by Cellosyl and mutanolysin, respectively, similarly revealed that both enzymes deacetylate GlcNAc residues of peptidoglycan. Kinetic analysis toward GlcNAc(2-6) revealed that GlcNAc4 was the favorable substrate for both enzymes. Identification of the sequence of N-acetychitooligosaccharides (GlcNAc(2-4)) following enzymatic deacetylation by using 1H NMR revealed that both enzymes deacetylate all GlcNAc residues of the oligomers except the reducing end ones. Enzymatic deacetylation of chemically acetylated vegetative peptidoglycan from B. cereus by BC1960 and BC3618 resulted in increased resistance to lysozyme digestion. This is the first biochemical study of bacterial peptidoglycan N-acetylglucosamine deacetylases.     

Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Delta5-desaturase from Phytophthora megasperma

The biosynthesis of arachidonic acid (20:4(Delta5Z,8Z,11Z,14Z)) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction. Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma. The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa. The desaturase sequence contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed. The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-gamma-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from omega3-arachidonic acid. Results presented here on the substrate specificity identify this expressed protein as a classical Delta5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Delta5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Delta8. Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine.     

Polyomavirus EGFP-pseudocapsids: analysis of model particles for introduction of proteins and peptides into mammalian cells

A vector for preparation of mouse polyomavirus capsid-like particles for transfer of foreign peptides or proteins into cells was constructed. Model pseudocapsids carrying EGFP fused with the C-terminal part of the VP3 minor protein (EGFP-VLPs) have been prepared and analysed for their ability to be internalised and processed by mouse cells and to activate mouse and human dendritic cells (DC) in vitro. EGFP-VLPs entered mouse epithelial cells, fibroblasts and human and mouse DC efficiently and were processed by both, lysosomes and proteasomes. Surprisingly, they did not induce upregulation of DC co-stimulation molecules or maturation markers in vitro; however, they did induce interleukin 12 secretion.     

Controlled reduction of the humidity induces a shortcut recovery reaction in the photocycle of photoactive yellow protein

The photocycle of the blue-light photoreceptor protein Photoactive Yellow Protein (PYP) was studied at reduced relative humidity (RH). Photocycle kinetics and spectra were measured in thin films of PYP in which the relative humidity was set at values between 29 and 98% RH with saturated solutions of various salts. We show that in this range, approximately 200 water molecules per PYP molecule are released from the film. As humidity decreased, photocycle transition rates changed, until at low humidity (RH < 50%) an authentic photocycle was no longer observed and the absorption spectrum of the dark, equilibrium state of PYP started to shift to 355 nm, that is, to a form resembling that of pB(dark). At moderately reduced humidity (i.e., >50% RH), an authentic photocycle is still observed, although its characteristics differ from those in solution. As humidity decreases, the rate of ground state recovery increases, while the rate of depletion of the first red-shifted intermediate pR dramatically decreases. The latter observation contrasts all so-far known modulations of the rate of the transition of the red-shifted- to the blue-shifted intermediates of PYP, which is consistently accelerated by all other modulations of the mesoscopic context of the protein. Under these same conditions, the long-lived, blue-shifted intermediate was formed not only with slower kinetics than in solution but also to a smaller extent. Global analysis of these data indicates that in this low humidity environment the photocycle can take a different route than in solution, that is, part of pG recovers directly from pR. These experiments on wild-type PYP, in combination with observations on a variant of PYP obtained by site-directed mutagenesis (the E46Q mutant protein), further document the context dependence of the photocycle transitions of PYP and are relevant for the interpretation of results obtained in both spectroscopic and diffraction studies with crystalline PYP.     

Photocycle of the flavin-binding photoreceptor AppA, a bacterial transcriptional antirepressor of photosynthesis genes

The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal domain belonging to a new class of photoreceptors designated BLUF domains. AppA was shown to control photosynthesis gene expression in response to blue light and oxygen tension. We have investigated the photocycle of the AppA BLUF domain by ultrafast fluorescence, femtosecond transient absorption, and nanosecond flash-photolysis spectroscopy. Time-resolved fluorescence experiments revealed four components of flavin adenine dinucleotide (FAD) excited-state decay, with lifetimes of 25 ps, 150 ps, 670 ps, and 3.8 ns. Ultrafast transient absorption spectroscopy revealed rapid internal conversion and vibrational cooling processes on excited FAD with time constants of 250 fs and 1.2 ps, and a multiexponential decay with effective time constants of 90 ps, 590 ps, and 2.7 ns. Concomitant with the decay of excited FAD, the rise of a species with a narrow absorption difference band near 495 nm was detected which spectrally resembles the long-living signaling state of AppA. Consistent with these results, the nanosecond flash-photolysis measurements indicated that formation of the signaling state was complete within the time resolution of 10 ns. No further changes were detected up to 15 micros. The quantum yield of the signaling-state formation was determined to be 24%. Thus, the signaling state of the AppA BLUF domain is formed on the ultrafast time scale directly from the FAD singlet excited state, without any apparent intermediate, and remains stable over 12 decades of time. In parallel with the signaling state, the FAD triplet state is formed from the FAD singlet excited state at 9% efficiency as a side reaction of the AppA photocycle.     

Photoisomerization and photoionization of the photoactive yellow protein chromophore in solution

Dispersed pump-dump-probe spectroscopy has the ability to characterize and identify the underlying ultrafast dynamical processes in complicated chemical and biological systems. This technique builds on traditional pump-probe techniques by exploring both ground- and excited-state dynamics and characterizing the connectivity between constituent transient states. We have used the dispersed pump-dump-probe technique to investigate the ground-state dynamics and competing excited-state processes in the excitation-induced ultrafast dynamics of thiomethyl p-coumaric acid, a model chromophore for the photoreceptor photoactive yellow protein. Our results demonstrate the parallel formation of two relaxation pathways (with multiple transient states) that jointly lead to two different types of photochemistry: cis-trans isomerization and detachment of a hydrated electron. The relative transition rates and quantum yields of both pathways have been determined. We find that the relaxation of the photoexcited chromophores involves multiple, transient ground-state intermediates and the chromophore in solution does not generate persistent photoisomerized products, but instead undergoes photoionization resulting in the generation of detached electrons and radicals. These results are of great value in interpreting the more complex dynamical changes in the optical properties of the photoactive yellow protein.     

De novo quantitative bisulfite sequencing using the pyrosequencing technology

Current protocols for DNA methylation analysis are either labor intensive or limited to the measurement of only one or two CpG positions. Pyrosequencing is a real-time sequencing technology that can overcome these limitations and be used as an epigenotype-mapping tool. Initial experiments demonstrated reliable quantification of the degree of DNA methylation when 2-6 CpGs were analyzed. We sought to improve the sequencing protocol so as to analyze as many CpGs as possible in a single sequencing run. By using an improved enzyme mix and adding single-stranded DNA-binding protein to the reaction, we obtained reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain good reproducibility and avoid preferential amplification. We applied the assay to the analysis of DNA methylation patterns in four CpG islands in the vicinity of IGF2 and H19 genes. This allowed accurate and quantitative de novo sequencing of the methylation state of each CpG, showing reproducible variations of methylation state in contiguous CpGs, and proved to be a useful adjunct to current technologies.