Bioluminescence reaction catalyzed by membrane-bound luciferase in the "firefly squid," Watasenia scintillans

The small Japanese "firefly squid," Watasenia scintillans, emits a bluish luminescence from dermal photogenic organs distributed along the ventral aspects of the head, mantle, funnel, arms and eyes. The brightest light is emitted by a cluster of three tiny organs located at the tip of each of the fourth pair of arms. Studies of extracts of the arm organs show that the light is due to a luciferin-luciferase reaction in which the luciferase is membrane-bound. The other components of the reaction are coelenterazine disulfate (luciferin), ATP, Mg(2+), and molecular oxygen. Based on the results, a reaction scheme is proposed which involves a rapid base/luciferase-catalyzed enolization of the keto group of the C-3 carbon of luciferin, followed by an adenylation of the enol group by ATP. The AMP serves as a recognition moiety for docking the substrate molecule to a luciferase bound to membrane, after which AMP is cleaved and a four-membered dioxetanone intermediate is formed by the addition of molecular oxygen. The intermediate then spontaneously decomposes to yield CO(2) and coelenteramide disulfate (oxyluciferin) in the excited state, which serves as the light emitter in the reaction.     

Bioluminescence of the arm light organs of the luminous squid Watasenia scintillans

The squid Watasenia scintillans emits blue light from numerous photophores. According to Tsuji [F.I. Tsuji, Bioluminescence reaction catalyzed by membrane-bound luciferase in the “firefly squid”, Watasenia scintillans, Biochim. Biophys. Acta 1564 (2002) 189–197.], the luminescence from arm light organs is caused by an ATP-dependent reaction involving Mg²⁺, coelenterazine disulfate (luciferin), and an unstable membrane-bound luciferase. We stabilized and partially purified the luciferase in the presence of high concentrations of sucrose, and obtained it as particulates (average size 0.6–2 µm). The ATP-dependent luminescence reaction of coelenterazine disulfate catalyzed by the particulate luciferase was investigated in detail. Optimum temperature of the luminescence reaction is about 5 °C. Coelenterazine disulfate is a strictly specific substrate in this luminescence system; any modification of its structure resulted in a very heavy loss in its light emission capability. The light emitter is the excited state of the amide anion form of coelenteramide disulfate. The quantum yield of coelenterazine disulfate is calculated at 0.36. ATP could be replaced by ATP-γ-S, but not by any other analogues tested. The amount of AMP produced in the luminescence reaction was much smaller than that of coelenteramide disulfate, suggesting that the reaction mechanism of the Watasenia bioluminescence does not involve the formation of adenyl luciferin as an intermediate.     

Role of molecular oxygen in the bioluminescence of the firefly squid, Watasenia scintillans

The bioluminescence system in the "firefly squid," Watasenia scintillans, is described. The light-emitting components consist of luciferin (coelenterazine disulfate), a membrane-bound luciferase, ATP, Mg2+, and molecular oxygen. A hypothetical scheme is proposed for the light-emitting reaction.     

Dimethyl-and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase

The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambda(em) = 639 nm. At higher pH values an additional emission maximum appears at lambda(em) = 500 nm (wavelength of excitation maximum lambda(ex) = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambda(abs) = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambda(abs) = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambda(abs) = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambda(ex) = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.     

Quenching of the fluorescence of Tyr and Trp residues of firefly luciferase from <Emphasis Type="Italic">Luciola mingrelica</Emphasis> by the substrates

Luciferase of the firefly Luciola mingrelica is characterized by fluorescence of not only the unique Trp residue (lambda(em) = 340 nm), but also that of Tyr residues (lambda(em) = 308 nm). Quenching of the intrinsic fluorescence of the luciferase by its substrates luciferin and ATP (AMP) has been studied. Luciferin (LH2) quenches Trp fluorescence more efficiently than the fluorescence of Tyr residues. Two centers of quenching of Tyr fluorescence by ATP have been found corresponding apparently to the allosteric and active sites of the luciferase with K(s(ATP)) = 20 and 110 microM, respectively. The influence of one substrate on the affinity of luciferase to the second was investigated using fluorescence. ATP (AMP) binding to the allosteric sites of the luciferase significantly affects the affinity of luciferase to LH2. Formation of the complex between the luciferase and LH2 affects the affinity of both allosteric and active sites of the luciferase to ATP (AMP). The observed effects are probably connected with conformational changes in the luciferase molecule upon its interaction with the substrates.     

A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.     

Transcription of a “silent” cyanobacterial psbA gene is induced by microaerobic conditions

Cyanobacteria, contrary to higher plants, have a small psbA gene family encoding the reaction centre D1 protein subunit of photosystem II, the first macromolecular pigment-protein complex of the photosynthetic electron transport chain. Modulation of expression of multiple psbA genes in the family allows cyanobacteria to adapt to changing environmental conditions. To date, two different strategies for regulation of the psbA genes have emerged. One, characterized in Synechocystis PCC6803 and Gloeobacter violaceus PCC7421 involves the increased expression of one type of D1 protein to cope with the increased rate of damage. The other strategy, in Synechococcus PCC7942 and Anabaena PCC7120, is to replace the existing D1 with a new D1 form for the duration of the stress. However, most of the psbA gene families characterized to date contain also a divergent, apparently silent psbA gene of unknown function. This gene, present in Synechocystis, Anabaena and Thermosynechococcus elongatus BP-1 was not induced by any stress condition applied so far. Our data shows a reversible induction of the divergent psbA gene during the onset of argon-induced microaerobic conditions in Synechocystis, Anabaena and Thermosynechococcus elongatus. The unitary functional response of three unrelated cyanobacterial species, namely the induction of the expression of the divergent psbA gene as a reaction to the same environmental cue, indicates that these genes and the protein they encode are part of a specific cellular response to microaerobic conditions. There are no specific primary structure similarities between the different microaerobic inducible D1 forms, designated as D1′. Only three amino acid residues are consistently conserved in D1′. These modifications are: G80 to A, F158 to L and T286 to L. In silico mutation of the published D1 structure from Thermosynechococcus did not reveal major modifications. The point by point effects of the mutations on the local environment of the PSII structure are also discussed.     

Relationship between stability and flexibility in the most flexible region of Photinus pyralis luciferase

Firefly luciferase is a protein with a large N-terminal and a small C-terminal domain. B-factor analysis shows that its C-terminal is much more flexible than its N-terminal. Studies on hyperthermophile proteins have been shown that the increased thermal stability of hyperthermophile proteins is due to their enhanced conformational rigidity and the relationship between flexibility, stability and function in most of proteins is on debate. Two mutations (D474K and D476N) in the most flexible region of firefly luciferase were designed. Thermostability analysis shows that D476N mutation doesn't have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became much more flexible than wild type although D476N doesn't have any significant difference. Intrinsic and ANS fluorescence studies demonstrate that D476N mutation is brought about by structural changes without significant effect on thermostability and flexibility. Molecular modeling reveals that disruption of a salt bridge between D⁴⁷⁴ and K⁴⁴⁵ accompanying with some H-bond deletion may be involved in destabilization of D474K mutant.     

Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: A membrane-associated “siderosome”

The peptidic siderophore pyoverdine is the primary iron uptake system of fluorescent pseudomonads, and a virulence factor in the opportunistic pathogen Pseudomonas aeruginosa. Pyoverdine biogenesis is a co-ordinate process requiring several precursor-generating enzymes and large nonribosomal peptide synthetases (NRPSs) in the cytoplasm, followed by extracytoplasmic maturation. By using cell fractionation, protein–protein interaction, and in vivo labeling assays we obtained evidence that, in P. aeruginosa, pyoverdine NRPSs assemble with precursor-generating enzymes into a membrane-bound multi-enzymatic complex, for which we propose the name “siderosome”. The pyoverdine biogenetic complex represents a novel example of subcellular compartmentalization of a secondary metabolic pathway in prokaryotes.     

A trip in the “New Microbiology” with the bacterial pathogen Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen causing an opportunistic disease called listeriosis. This bacterium invades and replicates in most cell types, due to its multiple strategies to exploit host molecular mechanisms. Research aiming at unravelling Listeria invasion and intracellular lifestyle has led to a number of key discoveries in infection biology, cell biology and also microbiology. In this review, we report on our most recent advances in understanding the intimate crosstalk between the bacterium and its host, resulting from in-depth studies performed over the past five years. We specifically highlight new concepts in RNA-based regulation in bacteria and discuss important findings in cell biology, including a new role for clathrin and an atypical mitochondrial fragmentation mechanism. We also illustrate the notion that bacterial infection regulates host gene expression at the chromatin level, contributing to an emerging field called patho-epigenetics. This review corresponds to the lecture given by one of us (P.C.) on the occasion of the 2014 FEBS|EMBO Woman in Science Award.     

The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of “upstream” Planar Cell Polarity proteins

The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.     

Molecular basis of diseases caused by the mtDNA mutation m.8969G>A in the subunit a of ATP synthase

The ATP synthase which provides aerobic eukaryotes with ATP, organizes into a membrane-extrinsic catalytic domain, where ATP is generated, and a membrane-embedded F_O domain that shuttles protons across the membrane. We previously identified a mutation in the mitochondrial MT-ATP6 gene (m.8969G>A) in a 14-year-old Chinese female who developed an isolated nephropathy followed by brain and muscle problems. This mutation replaces a highly conserved serine residue into asparagine at amino acid position 148 of the membrane-embedded subunit a of ATP synthase. We showed that an equivalent of this mutation in yeast (aS₁₇₅N) prevents F_O-mediated proton translocation. Herein we identified four first-site intragenic suppressors (aN₁₇₅D, aN₁₇₅K, aN₁₇₅I, and aN₁₇₅T), which, in light of a recently published atomic structure of yeast F_O indicates that the detrimental consequences of the original mutation result from the establishment of hydrogen bonds between aN₁₇₅ and a nearby glutamate residue (aE₁₇₂) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS₁₇₅N mutation can be suppressed by second-site suppressors (aP₁₂S, aI₁₇₁F, aI₁₇₁N, aI₂₃₉F, and aI₂₀₀M), of which some are very distantly located (by 20–30?Å) from the original mutation. The possibility to compensate through long-range effects the aS₁₇₅N mutation is an interesting observation that holds promise for the development of therapeutic molecules.     

A straightforward kinetic evidence for coexistence of “induced fit” and “selected fit” in the reaction mechanism of a mutant tryptophan indole lyase Y72F from Proteus vulgaris

The interaction of the mutant tryptophan indole-lyase (TIL) from Proteus vulgaris Y72F with the transition state analogue, oxindolyl-l-alanine (OIA), with the natural substrate, l-tryptophan, and with a substrate S-ethyl-l-cysteine was examined. In the case of wild-type enzyme these reactions are described by the same kinetic scheme where binding of holoenzyme with an amino acid, leading to reversible formation of an external aldimine, proceeds very fast, while following transformations, leading finally to reversible formation of a quinonoid intermediate proceed with measureable rates. Principally the same scheme (“induced fit”) is realized in the case of mutant Y72F enzyme reaction with OIA. For the reaction of mutant enzyme with l-Trp at lower concentrations of the latter a principally different kinetic scheme is observed. This scheme suggests that binding of the substrate and formation of the quinonoid intermediate are at fast equilibrium, while preceding conformational changes of the holoenzyme proceed with measureable rates (“selected fit”). For the reaction with S-ethyl-l-cysteine the observed concentration dependence of k_obs agrees with the realization of both kinetic schemes, the “selected fit” becoming predominant at lower concentrations of substrate, the “induced fit”— at higher ones. In the reaction with S-ethyl-l-cysteine the formation of the quinonoid intermediate proceeds slower than does catalytic α,β-elimination of ethylthiol from S-ethyl-l-cysteine, and consequently does not play a considerable role in the catalysis, which may be effected by a concerted E2 mechanism.     

Synthesis, spectroscopic and structural characterization of the high-spin Fe(II) cyanato-N and thiocyanato-N “picket fence” porphyrin complexes

Two new iron(II) five-coordinated porphyrin complexes [Na(2,2,2-crypt)] [Fe^II(TpivPP)(NCO)] (1) (TpivPP = α,α,α,α-tetrakis(o-pivalamidophenyl) porphyrin known as picket fence porphyrin and 2,2,2-crypt is the cryptand-222) and [K(2,2,2-crypt)][Fe^II(TpivPP)(NCS)] (2) have been prepared and characterized. The UV-Vis and IR spectroscopic data are consistent with a cyanato-N and thiocyanato-N ferrous porphyrinates. The Mössbauer data and the X-ray structural analysis indicate that the Fe(II) cation in 1 and 2 is high-spin (S = 2) and has the (d_xy)²(d_xz)¹(d_yz)¹(d_z²)¹(d_x²-y²)¹ ground state electronic configuration.For complex 1, the average equatorial iron-pyrrole N bond length (Fe-N_p = 2.120(2) Å), the distance between the iron and the 24-atom mean plane of the porphyrin ring (Fe-P_C = 0.6805(7) Å) and the distance between the iron and the plane made by the four pyrrole nitrogens (Fe-P_N = 0.5923(12) Å) are longer than those of complex 2 and similar five-coordinated Fe(II) high-spin porphyrinates. This is probably due to the significant electronic repulsion of the d_x²-y² and d_xy orbitals by the negative charge of the pyrrole N atoms in case of 1.     

Taxonomical status and phylogenetic relations between the “thomasi” and “atticus” chromosomal races of the underground vole Microtus thomasi (Rodentia, Arvicolinae)

Laboratory crosses among wild caught individuals of the chromosomal races “atticus” and “thomasi”, were performed to analyze the degree of interracial reproductive isolation. The fertility of the studied specimens was evaluated by taking into consideration the reproductive success, the litter size and performing comparative histological examination of the testicular material. All studied populations were submitted to classical cytogenetic and mitochondrial analysis (cytochrome b gene), providing new evidences to the potential phylogenetic relations and taxonomical status of the two chromosomal races. The previously described “atticus” populations are divided in two genetically distinct, geographically and reproductively isolated lineages (2.9% total and 2.4% net divergence), which probably derived from different glacial refugia of Southern Greece. Here, we suggest that the lineage, consisting of the populations from Attiki and Evia Island, should be distinguished as a valid species, named Microtus atticus, including the two chromosomal races “atticus” and “evia”. On the contrary, the ex-“atticus” populations from North Peloponnesus belong to the same mitochondrial lineage with the other Microtus thomasi populations and should be considered as a chromosomal polymorphism inside the chromosomal race “thomasi”.