Biomedical subjects

K J Hellingwerf

Publications and source records attributed to K J Hellingwerf.

At least 55 records · Page 3Linked to original sources

PubMed1996

Glu46 donates a proton to the 4-hydroxycinnamate anion chromophore during the photocycle of photoactive yellow protein.

Photoactive yellow protein (PYP) is a photoreceptor containing a unique 4-hydroxycinnamic acid (pCA) chromophore. The trans to cis photoisomerization of this chromophore activates a photocycle involving first a short-lived red-shifted intermediate (pR), then a long-lived blue-shifted intermediate (pB), and finally recovery of the original receptor state (pG). The pCA chromophore is deprotonated in pG and protonated in pB, but the proton donor for this process has not yet been identified. Here we report the first FTIR spectroscopic data on pG, pR, and pB. The IR difference signals in the carbonyl stretching region of COOH groups (1700-1800 cm-1) reveal that a buried carboxylic group close to the chromophore (i) is protonated in pG, (ii) develops a stronger hydrogen bonding in pR, and (iii) becomes deprotonated in pB. These signals are unambiguously assigned to Glu46, on the basis of the IR data and the 1.4 A X-ray structure of PYP [Borgstahl et al. (1995) Biochemistry 34, 6278-6287]. Our data demonstrate that in pR Glu46 remains in hydrogen bonding contact with the negatively charged phenolic oxygen of pCA after chromophore photoisomerization. This strongly implies that the chromophore is isomerized to the 7-cis 9-s-trans conformation in pR, resulting from co-isomerization of both the C7 = C8 and C9-C10 bonds. In the pR to pB transition, Glu46 becomes deprotonated, concomitant with chromophore protonation. Therefore, we conclude that Glu46 functions as the proton donor for the protonation of pCA during the PYP photocycle. We propose a molecular mechanism in which intramolecular proton transfer in PYP leads to global protein conformational changes involved in signal transduction.

Bacterial Proteins↗

PubMed1996

The xanthopsins: a new family of eubacterial blue-light photoreceptors.

Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic phototrophic purple bacteria. The PYP from Ectothiorhodospira halophila BN9626 is the only member for which the sequence has been reported at the DNA level. Here we describe the cloning and sequencing of the genes encoding the PYPs from E.halophila SL-1 (type strain) and Rhodospirillum salexigens. The latter protein contains, like the E.halophila PYP, the chromophore trans p-coumaric acid, as we show here with high performance capillary zone electrophoresis. Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well-characterized bacterium in which this photoreceptor has been identified. An ORF downstream of the pyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis of the chromophore of PYP. The pyp gene from E.halophila was used for heterologous overexpression in both Escherichia coli and R.sphaeroides, aimed at the development of a holoPYP overexpression system (an intact PYP, containing the p-coumaric acid chromophore and displaying the 446 nm absorbance band). In both organisms the protein could be detected immunologically, but its yellow color was not observed. Molecular genetic construction of a histidine-tagged version of PYP led to its 2500-fold overproduction in E.coli and simplified purification of the heterologously produced apoprotein. HoloPYP could be reconstituted by the addition of p-coumaric anhydride to the histidine-tagged apoPYP (PYP lacking its chromophore). We propose to call the family of photoactive yellow proteins the xanthopsins, in analogy with the rhodopsins.

Amino Acid Sequence↗

PubMed1996

Evidence for trans-cis isomerization of the p-coumaric acid chromophore as the photochemical basis of the photocycle of photoactive yellow protein.

Analysis of the chromophore p-coumaric acid, extracted from the ground state and the long-lived blue-shifted photocycle intermediate of photoactive yellow protein, shows that the chromophore is reversibly converted from the trans to the cis configuration, while progressing through the photocycle. The detection of the trans and cis isomers was carried out by high performance capillary zone electrophoresis and further substantiated by 1H NMR spectroscopy. The data presented here establish the photo-isomerization of the vinyl double bond in the chromophore as the photochemical basis for the photocycle of photoactive yellow protein, a eubacterial photosensory protein. A similar isomerization process occurs in the structurally very different sensory rhodopsins, offering an explanation for the strong spectroscopic similarities between photoactive yellow protein and the sensory rhodopsins. This is the first demonstration of light-induced isomerization of a chromophore double bond as the photochemical basis for photosensing in the domain of Bacteria.

Bacterial Proteins↗

PubMed1996

Chemical reactivity and spectroscopy of the thiol ester-linked p-coumaric acid chromophore in the photoactive yellow protein from Ectothiorhodospira halophila.

We have recently identified p-coumaric acid as the chromophore of the photoactive yellow protein (PYP) from the purple sulfur bacterium Ectothiorhodospira halophila, a blue-light photoreceptor with rhodopsin-like photochemistry [Hoff, W. D., Düx, P., Hård, K., Nugteren-Roodzant, I. M., Crielaard, W., Boelens, R., Kaptein, R., Van Beeumen, J., & Hellingwerf, K. J. (1994) Biochemistry 33, 13959-13962]. Here we report on the chemistry of the linkage of this new photoactive cofactor to apoPYP: (i) Analysis of chromophore-peptide conjugates of PYP by high-resolution mass spectrometry unambiguously shows that the p-coumaric acid molecule is bound to Cys 69 via a thiol ester bond. The PYP chromophore is the first cofactor known to be stably thiol ester-linked to its apoprotein. (ii) The chemical reactivity of this thiol ester bond with respect to dithiothreitol, performic acid, and high pH is similar to that of disulfide bridges. These treatments result in the cleavage of the thiol ester bond, concomitant with strong shifts in the UV/vis absorbance band of the chromophore. (iii) The spectral properties of the PYP chromophore under different conditions are related to the structural integrity of the protein, the presence of the thiol ester bond, and the ionization state of the phenolic proton of the chromophore. These results are important for the general problem of spectral tuning in photoreceptor proteins.

Amino Acid Sequence↗

PubMed1996

Protein folding thermodynamics applied to the photocycle of the photoactive yellow protein.

Two complementary aspects of the thermodynamics of the photoactive yellow protein (PYP), a new type of photoreceptor that has been isolated from Ectothiorhodospira halophila, have been investigated. First, the thermal denaturation of PYP at pH 3.4 has been examined by global analysis of the temperature-induced changes in the UV-VIS absorbance spectrum of this chromophoric protein. Subsequently, a thermodynamic model for protein (un)folding processes, incorporating heat capacity changes, has been applied to these data. The second aspect of PYP that has been studied is the temperature dependence of its photocycle kinetics, which have been reported to display an unexplained deviation from normal Arrhenius behavior. We have extended these measurements in two solvents with different hydrophobicities and have analyzed the number of rate constants needed to describe these data. Here we show that the resulting temperature dependence of the rate constants can be quantitatively explained by the application of a thermodynamic model which assumes that heat capacity changes are associated with the two transitions in the photocycle of PYP. This result is the first example of an enzyme catalytic cycle being described by a thermodynamic model including heat capacity changes. It is proposed that a strong link exists between the processes occurring during the photocycle of PYP and protein (un)folding processes. This permits a thermodynamic analysis of the light-induced, physiologically relevant, conformational changes occurring in this photoreceptor protein.

1-Butanol↗

PubMed1996

Photobiology of microorganisms: how photosensors catch a photon to initialize signalling.

Photobiological processes are relevant for microorganisms for energy generation, protection against excess and/or damaging radiation, and for signalling. In this review we give an overview of the knowledge on the functioning of photosensors in microorganisms, with special emphasis on the conformational changes that lead to signal generation and transduction. Light is absorbed by specific chromophores, which are tuned, by their proteinaceous environment, to function optimally. These chromophores belong to three classes: tetrapyrroles, polyenes and aromatics. The chemical structure of photosensing pigment/protein complexes has been resolved for many of the photobiological processes that have a characteristic sensitivity in the visible and infrared part of the spectrum of (solar) radiation. However, knowledge about the structure of photoreceptors responsible for several physiologically well-characterized photoresponses to UV- and blue light is still lacking. For a limited number of phototransduction processes, the details of light-induced signal transfer are beginning to be understood in atomic detail. This applies particularly to two photosensors involved in phototactic responses in bacteria: sensory rhodopsin I (SR-I) from Halobacterium salinarium and photoactive yellow protein (PYP) from Ectothiorhodospira halophila. The SR-1 system is of special interest because the transducer accepting the signal from SR-1 was recently identified as Htr-1, a homologue of the methyl-accepting chemotaxis proteins which have been characterized in Escherichia coli. PYP, on the other hand, may be the first photosensor to actually reveal all relevant details of the kinetics, thermodynamics, and molecular motion of light-induced signal generation, through an understanding of how the photo-isomerization of the chromophore forces the sensor protein into the signalling state. Here we compare these photosensors and discuss common themes in the initiation of photosensory signal transduction in microorganisms in terms of the molecular properties of photosensors and their signalling state.

Bacteria↗

PubMed1996

Specific detection and analysis of a probiotic Bifidobacterium strain in infant feces.

For specific detection of the probiotic Bifidobacterium sp. strain LW420 in infant feces and for rapid quality control of this strain in culture, three strain-specific 16S rRNA gene-targeted primers have been developed. These primers allow specific detection of the organism via PCR. Specificity of the primers was determined in DNA samples isolated from single-strain and mixed cultures of bifidobacteria and in heterogenous fecal samples. The feasibility of this method for use in specific detection of probiotic strains was investigated through addition of Bifidobacterium sp. strain LW420 to infant instant milk formula (IMF) and PCR analyses of bacterial DNA isolated from feces of 17 newborn IMF-fed infants. In feces of all nine babies that had been fed with the probiotic IMF, the strain-specific PCR signal could be detected. No signal was found in feces of the eight infants that had been fed with a nonprobiotic IMF, demonstrating the specificity of the PCR method. All 17 infants developed a major fecal Bifidobacterium population already after 3 days, as determined through genus-specific and strain-specific PCR. Phenotypical screening of Bifidobacterium sp. strain LW420 and analysis of homology of the 16S rRNA gene sequence of this strain with that of other bifidobacteria deposited in databases do not allow positive classification of LW420 among the currently known species of Bifidobacterium.

Animals↗

PubMed1996

Physiological factors affecting production of extracellular lipase (LipA) in Acinetobacter calcoaceticus BD413: fatty acid repression of lipA expression and degradation of LipA.

The extracellular lipase (LipA) produced by Acinetobacter calcoaceticus BD413 is required for growth of the organism on triolein, since mutant strains that lack an active lipase fail to grow with triolein as the sole carbon source. Surprisingly, extracellular lipase activity and expression of the structural lipase gene (lipA), the latter measured through lacZ as a transcriptional reporter, are extremely low in triolein cultures of LipA+ strains. The explanation for this interesting paradox lies in the effect of fatty acids on the expression of lipA. We found that long-chain fatty acids, especially, strongly repress the expression of lipA, thereby negatively influencing the production of lipase. We propose the involvement of a fatty acyl-responsive DNA-binding protein in regulation of expression of the A. calcoaceticus lipBA operon. The potential biological significance of the observed physiological competition between expression and repression of lipA in the triolein medium is discussed. Activity of the extracellular lipase is also negatively affected by proteolytic degradation, as shown in in vitro stability experiments and by Western blotting (immunoblotting) of concentrated supernatants of stationary-phase cultures. In fact, the relatively high levels of extracellular lipase produced in the early stationary phase in media which contain hexadecane are due only to enhanced stability of the extracellular enzyme under those conditions. The rapid extracellular degradation of LipA of A. calcoaceticus BD413 by an endogenous protease is remarkable and suggests that proteolytic degradation of the enzyme is another important factor in regulating the level of active extracellular lipase.

Acinetobacter calcoaceticus↗

PubMed1996

Purification of a lipase from Acinetobacter calcoaceticus AAC323-1 by hydrophobic-interaction methods.

The performance of hydrophobic-interaction chromatography (HIC) for the purification of Acinetobacter calcoaceticus AAC323-1 lipase was compared with that of various aqueous two-phase systems. While a 42% lipase yield with a purification factor of 140 could be recovered by HIC, higher yields were achieved by using aqueous two-phase systems, either those formed by poly(ethylene glycol) and dextran or those based upon the use of a detergent. Triton X-114-based aqueous two-phase partition showed the best performance, with a yield of 81% and a purification factor of 68. Further detergent removal was easily achieved with an adsorbent, with no significant decrease in yields. Owing to its simplicity, the method should be easy to scale-up.

Acinetobacter calcoaceticus↗

PubMed1996

Quantitative conversion of glucose into glucose 6-phosphate by intact Escherichia coli cells.

The use of intact Escherichia coli cells for the conversion of glucose into glucose 6-phosphate using the Uhp system for transport of the phosphorylated sugar out of the cell was investigated. The strain E. coli DF214, which is not capable of glucose 6-phosphate catabolism via glycolysis or the Entner-Douderoff pathway, was used. The efflux of glucose 6-phosphate was dependent on the presence of UhpT, the hexose-phosphate transporter, plus the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone or the ionophore valinomycin. At low glucose concentrations (e.g. 2.5 mM), near-quantitative conversion (> or = 80%) of glucose into extracellular glucose 6-phosphate can be achieved. When cells are incubated for a shorter period of time, complete conversion can occur.

Escherichia coli↗

PubMed1995

Resonance Raman evidence that the thioester-linked 4-hydroxycinnamyl chromophore of photoactive yellow protein is deprotonated.

Resonance Raman spectra of the ground state of photoactive yellow protein (PYP), a photoactive pigment found in Ectothiorhodospira halophila, have been obtained with excitation at 413.1 nm using a microspinning sample cell. The resonance Raman spectra of the thioester-linked 4-hydroxycinnamyl chromophore in the protein are compared with the preresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and 4-hydroxycinnamic acid model compounds at various pH values. Bands at 1568, 1542, 1500, 1434, and 1166 cm-1 in the Raman spectrum of the anionic form of the 4-hydroxycinnamyl phenyl thioester are shown to be characteristic for the deprotonation of the chromophore. The observation of bands in PYP exhibiting very similar frequency and intensity patterns provides strong evidence that the chromophore in PYP is stabilized as a phenolate anion at pH 7.4, in support of conclusions from crystallographic studies. Furthermore, the insensitivity of the PYP Raman spectrum to placement of the protein in D2O buffer is consistent with the absence of the exchangeable phenolic proton on the cinnamyl chromophore. These results establish the feasibility of elucidating the molecular mechanism of light-to-information transduction by this new photosensory pigment with resonance Raman spectroscopy.

Bacterial Proteins↗

PubMed1995

Non-physiological expression of UhpT does not lead to uncontrolled leakage of sugar phosphates out of Escherichia coli cells.

De-regulated expression of uhpT under control of the tac promoter, by increasing concentrations of isopropyl-thio-beta-D-galactoside, progressively inhibited the growth rate of Escherichia coli cells, to such an extent that growth was fully inhibited at 1 mM of the inducer. Significantly, additio of glucose 6-phosphate to the growth medium of the cells did not protect against this inhibition. Furthermore, efflux of sugar phosphates from the cells did not take place under these conditions, unless protonophoric uncouplers were added. We therefore conclude that the regulation of uhpT expression, i.e. via extracellular induction through a two-component system, did not evolve in order to prevent loss of essential metabolites from the cytoplasm under conditions when extracellular sugar phosphates are not available.

Biological Transport↗

PubMed1995

Acinetobacter calcoaceticus liberates chromosomal DNA during induction of competence by cell lysis.

A transformation assay was used to assay the amount of DNA present in the extracellular medium of a growing culture of Acinetobacter calcoaceticus. It was observed that small amounts of DNA were liberated during the entire exponential growth phase in a batch culture. Release of DNA could be fully accounted for by lysis of cells. Lysis was quantified via simultaneous measurement of beta-galactosidase activity of cells and supernatant, with a strain that contained a plasmid (pAPA100) with lacZ under control of a constitutive beta-lactamase promoter. In conclusion, no evidence could be obtained indicating that Acinetobacter calcoaceticus actively excretes DNA, to be used for DNA exchange.

Acinetobacter calcoaceticus↗

PubMed1995

Photoinduced volume change and energy storage associated with the early transformations of the photoactive yellow protein from Ectothiorhodospira halophila.

The photocycle of the photoactive yellow protein (PYP) isolated from Ectothiorhodospira halophila was analyzed by flash photolysis with absorption detection at low excitation photon densities and by temperature-dependent laser-induced optoacoustic spectroscopy (LIOAS). The quantum yield for the bleaching recovery of PYP, assumed to be identical to that for the phototransformation of PYP (pG), to the red-shifted intermediate, pR, was phi R = 0.35 +/- 0.05, much lower than the value of 0.64 reported in the literature. With this value and the LIOAS data, an energy content for pR of 120 kJ/mol was obtained, approximately 50% lower than for excited pG. Concomitant with the photochemical process, a volume contraction of 14 ml/photoconverted mol was observed, comparable with the contraction (11 ml/mol) determined for the bacteriorhodopsin monomer. The contraction in both cases is interpreted to arise from a protein reorganization around a phototransformed chromophore with a dipole moment different from that of the initial state. The deviations from linearity of the LIOAS data at photon densities > 0.3 photons per molecule are explained by absorption by pG and pR during the laser pulse duration (i.e., a four-level system, pG, pR, and their respective excited states). The data can be fitted either by a simple saturation process or by a photochromic equilibrium between pG and pR, similar to that established between the parent chromoprotein and the first intermediate(s) in other biological photoreceptors. This nonlinearity has important consequences for the interpretation of the data obtained from in vitro studies with powerful lasers.

Bacterial Proteins↗

PubMed1995

Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene.

The extracellular lipase from Acinetobacter calcoaceticus BD413 was purified to homogeneity, via hydrophobic-interaction fast performance liquid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source. The enzyme has an apparent molecular mass of 32 kDa on SDS-polyacrylamide gels and hydrolyses long acyl chain p-nitrophenol (pNP) esters, like pNP palmitate (pNPP), with optimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and towards egg-yolk emulsions. The N-terminal amino acid sequence of the mature protein was determined, and via reverse genetics the structural lipase gene was cloned from a gene library of A. calcoaceticus DNA in Escherichia coli phage M13. Sequence analysis of a 2.1 kb chromosomal DNA fragment revealed one complete open reading frame, lipA, encoding a mature protein with a predicted molecular mass of 32.1 kDa. This protein shows high similarity to known lipases, especially Pseudomonas lipases, that are exported in a two-step secretion mechanism and require a lipase-specific chaperone. The identification of an export signal sequence at the N-terminus of the mature lipase suggests that the lipase of Acinetobacter is also exported via a two-step translocation mechanism. However, no chaperone-encoding gene was found downstream of lipA, unlike the situation in Pseudomonas. Analysis of an A. calcoaceticus mutant showing reduced lipase production revealed that a periplasmic disulphide oxidoreductase is involved in processing of the lipase. Via sequence alignments, based upon the crystal structure of the closely related Pseudomonas glumae lipase, a model has been made of the secondary-structure elements in AcLipA. The active site serine of AcLipA was changed to an alanine, via site-directed mutagenesis, resulting in production of an inactive extracellular lipase.

Acinetobacter calcoaceticus↗

PubMed1995

Signal transduction in bacteria: phospho-neural network(s) in Escherichia coli?

The molecular basis of many forms of signal transfer in living organisms is provided via the transient phosphorylation of regulatory proteins by transfer of phosphoryl groups between these proteins. The dominant form of signal transduction in prokaryotic microorganisms proceeds via so-called two-component regulatory systems. These systems constitute phosphoryl transfer pathways, consisting of two or more components. Most of these pathways are linear, but some converge and some are divergent. The molecular properties of some of the well-characterised representatives of two-component systems comply with the requirements to be put upon the elements of a neural network: they function as logical operators and show the phenomenon of autoamplification. Because there are many phosphoryl transfer pathways in parallel and because there also appears to be cross-talk between these pathways, the total of all two-component regulatory systems in a single prokaryotic cell may show the typical characteristics of a 'phospho-neural network'. This may well lead to signal amplification, associative responses and memory effects, characteristics which are typical for neural networks. One of the main challenges in molecular microbial physiology is to determine the extent of the connectivity of the constituting elements of this presumed 'phospho-neural network', and to outline the extent of intelligence-like behaviour this network can generate. Escherichia coli is the organism of choice for this characterization.

Escherichia coli↗

PubMed1995

Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase.

Acinetobacter calcoaceticus BD413 produces an extracellular lipase, which is encoded by the lipA gene. Five lipase-deficient mutants have been generated via random insertion mutagenesis. Phenotypic characterization of these mutants revealed the presence of as many as four lipolytic enzymes in A. calcoaceticus. Biochemical evidence classified four of the mutants as export mutants, which presumably are defective in translocation of the lipase across the outer membrane. The additional mutant, designated AAC302, displays a LipA- phenotype, and yet the mutation in this strain was localized 0.84 kbp upstream of lipA. Sequence analysis of this region revealed an open reading frame, designated lipB, that is disrupted in AAC302. The protein encoded by this open reading frame shows extensive similarity to a chaperone-like helper protein of several pseudomonads, required for the production of extracellular lipase. Via complementation of AAC302 with a functional extrachromosomal copy of lipA, it could be determined that LipB is essential for lipase production. As shown by the use of a translational LipB-PhoA fusion construct, the C-terminal part of LipB of A. calcoaceticus BD413 is located outside the cytoplasm. Sequence analysis further strongly suggests that A. calcoaceticus LipB is N terminally anchored in the cytoplasmic membrane. Therefore, analogous to the situation in Pseudomonas species, however, lipB in A. calcoaceticus is located upstream of the structural lipase gene. lipB and lipA form a bicistronic operon, and the two genes are cotranscribed from an Escherichia coli sigma 70-type promoter. The reversed order of genes, in comparison with the situation in Pseudomonas species, suggests that LipA and LipB are produced in equimolar amounts. Therefore, the helper protein presumably does not only have a catalytic function, e.g., in folding of the lipase, but is also likely to act as a lipase-specific chaperone. A detailed model of the export route of the lipase of A. calcoaceticus BD413 is proposed.

Acinetobacter calcoaceticus↗

PubMed1994

Thiol ester-linked p-coumaric acid as a new photoactive prosthetic group in a protein with rhodopsin-like photochemistry.

A number of Eubacteria contain a photoactive yellow protein which has a photosensory function in negative phototaxis. It has been proposed that the cofactor responsible for the intense yellow color of this protein is retinal [McRee, D. E., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6533-6537]. This would make it the first eubacterial rhodopsin. Here we report the chemical structure of this chromophoric group to be p-coumaric acid, which is covalently bound to a unique cysteine in the apoprotein via a thiol ester bond, and thus not retinal. This makes PYP the first example of a protein containing p-coumaric acid, a metabolite previously found only in plants, as a prosthetic group and establishes the photoactive yellow proteins as a new type of photochemically active receptor molecule.

Chromatiaceae↗