Design and Signaling Mechanism of Light-Regulated Histidine Kinases

Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by ∼ 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40°-60° rotational movement within an α-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by α-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.     

Structural basis for light-dependent signaling in the dimeric LOV domain of the photosensor YtvA

The photosensor YtvA binds flavin mononucleotide and regulates the general stress reaction in Bacillus subtilis in response to blue light illumination. It belongs to the family of light-oxygen-voltage (LOV) proteins that were first described in plant phototropins and form a subgroup of the Per-Arnt-Sim (PAS) superfamily. Here, we report the three-dimensional structure of the LOV domain of YtvA in its dark and light states. The protein assumes the global fold common to all PAS domains and dimerizes via a hydrophobic interface. Directly C-terminal to the core of the LOV domain, an alpha-helix extends into the solvent. Light absorption causes formation of a covalent bond between a conserved cysteine residue and atom C(4a) of the FMN ring, which triggers rearrangements throughout the LOV domain. Concomitantly, in the dark and light structures, the two subunits of the dimeric protein rotate relative to each other by 5 degrees . This small quaternary structural change is presumably a component of the mechanism by which the activity of YtvA is regulated in response to light. In terms of both structure and signaling mechanism, YtvA differs from plant phototropins and more closely resembles prokaryotic heme-binding PAS domains.     

Vibrational spectroscopy of an algal Phot-LOV1 domain probes the molecular changes associated with blue-light reception

The LOV1 domain of the blue light Phot1-receptor (phototropin homolog) from Chlamydomonas reinhardtii has been studied by vibrational spectroscopy. The FMN modes of the dark state of LOV1 were identified by preresonance Raman spectroscopy and assigned to molecular vibrations. By comparing the blue-light-induced FTIR difference spectrum with the preresonance Raman spectrum, most of the differences are due to FMN modes. Thus, we exclude large backbone changes of the protein that might occur during the phototransformation of the dark state LOV1-447 into the putative signaling state LOV1-390. Still, the presence of smaller amide difference bands cannot be excluded but may be masked by overlapping FMN modes. The band at 2567 cm(-1) is assigned to the S-H stretching vibration of C57, the residue that forms the transient thio-adduct with the chromophore FMN. The occurrence of this band is evidence that C57 is protonated in the dark state of LOV1. This result challenges conclusions from the homologous LOV2 domain from oat that the thiolate of the corresponding cysteine is the reactive species.     

The Switch that Does Not Flip: The Blue-Light Receptor YtvA from Bacillus subtilis Adopts an Elongated Dimer Conformation Independent of the Activation State as Revealed by a Combined AUC and SAXS Study

Photoreceptors play an important role in plants and bacteria by converting extracellular stimuli into intracellular signals. One distinct class are the blue-light-sensitive phototropins harboring a light-oxygen-voltage (LOV) domain coupled to various effector domains. Photon absorption by the chromophore within the LOV domain results in an activation of the output domain via mechanisms that are hitherto not well understood. The photoreceptor YtvA from Bacillus subtilis is a bacterial analog of phototropins, consists of an LOV and a sulfate transporter/anti-sigma factor antagonist domain, and is involved in the response of the bacterium to environmental stress. We present here analytical ultracentrifugation studies and small-angle X-ray scattering experiments, showing that YtvA is a dimer. On the basis of these results, we present a low-resolution model of the dimer in the dark and the lit state of the protein. In addition, we show that YtvA does not change its oligomerization state or its overall shape upon light activation.     

Molecular determinants of dihydrouridine synthase activity

Dihydrouridine is one of the most abundant modified bases in tRNA. However, little is known concerning the biochemistry of dihydrouridine synthase (DUS) enzymes. To identify molecular determinants that are necessary for DUS activity, we have developed a DUS-complementation assay in Escherichia coli. Using this assay, we have identified amino-acid residues that are critical for the activity of YjbN, an E. coli DUS. We also show that the aq1598 gene product, a putative DUS from Aquifex aeolicus, catalyzes dihydrouridine formation, providing the first biochemical demonstration that A. aeolicus encodes an active DUS.     

Crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis

With the aim of elucidating the biological function of hypothetical proteins unique amongst the Actynomyces sub-group of bacteria, we have solved the crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis at 1.8 A resolution. Rv1155 is a homodimer both in the crystal structure and in solution and folds into two separate domains consisting of a six-stranded anti-parallel beta-barrel fold flanked by two alpha-helices and a helix-turn-helix domain. Both domains contribute to the formation of two deep clefts at the dimer interface. The overall fold of Rv1155 strikingly resembles that of flavin mononucleotide-binding protein and pyridoxamine 5'-phosphate oxydase, but the architecture of the putative binding pocket is markedly different, consistent with the lack of color of Rv1155 and its inability to bind FMN. Rv1155 thus appears to belong to a group of proteins with stringent conservation of the binding cleft, having evolved towards a new binding function.     

Crystal structure of the flavoprotein ArsH from Sinorhizobium meliloti

Purified ArsH from Sinorhizobium meliloti exhibits NADPH:FMN-dependent reduction of molecular O2 to hydrogen peroxide and catalyzes reduction of azo dyes. The structure of ArsH was determined at 1.8A resolution. ArsH crystallizes with eight molecules in the asymmetric unit forming two tetramers. Each monomer has a core domain with a central five-stranded parallel beta-sheet and two monomers interact to form a classical flavodoxin-like dimer. The N- and C-terminal extensions of ArsH are involved in interactions between subunits and tetramer formation. The structure may provide insight in how ArsH participates in arsenic detoxification.     

Catalytic role of a conserved cysteine residue in the desulfonation reaction by the alkanesulfonate monooxygenase enzyme

Detailed kinetic studies were performed in order to determine the role of the single cysteine residue in the desulfonation reaction catalyzed by SsuD. Mutation of the conserved cysteine at position 54 in SsuD to either serine or alanine had little effect on FMNH₂ binding. The k_cat/K_m value for the C54S SsuD variant increased 3-fold, whereas the k_cat/K_m value for C54A SsuD decreased 6-fold relative to wild-type SsuD. An initial fast phase was observed in kinetic traces obtained for the oxidation of flavin at 370 nm when FMNH₂ was mixed against C54S SsuD (k_obs, 111 s^− 1) in oxygenated buffer that was 10-fold faster than wild-type SsuD (k_obs, 12.9 s^− 1). However, there was no initial fast phase observed in similar kinetic traces obtained for C54A SsuD. This initial fast phase was previously assigned to the formation of the C4a-(hydro)peroxyflavin in studies with wild-type SsuD. There was no evidence for the formation of the C4a-(hydro)peroxyflavin with either SsuD variant when octanesulfonate was included in rapid reaction kinetic studies, even at low octanesulfonate concentrations. The absence of any C4a-(hydro)peroxyflavin accumulation correlates with the increased catalytic activity of C54S SsuD. These results suggest that the conservative serine substitution is able to effectively take the place of cysteine in catalysis. Conversely, decreased accumulation of the C4a-(hydro)peroxyflavin intermediate with the C54A SsuD variant may be due to decreased activity. The data described suggest that Cys54 in SsuD may be either directly or indirectly involved in stabilizing the C4a-(hydro)peroxyflavin intermediate formed during catalysis through hydrogen bonding interactions.     

Inhibition of Photosystem II by uncouplers at alkaline pH and its reversal by artificial electron donors

When chloroplasts are aged for 5 min at pH 9.6, or are exposed to uncouplers at pH 8.5–9.0, electron flow from water to Hill acceptors is inhibited. Both treatments induce rapid millisecond dark decay of delayed light emission. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-sensitive electron transport through Photosystem II can be regenerated in both types of inhibited chloroplasts by the artificial electron donor, 1,5-diphenylcarbohydrazide. Neither treatment inhibits electron flow through Photosystem I. Uncouplers at alkaline pH, when added in the light, are less effective in producing the inhibition than when added in the dark. These results are interpreted as indicating inhibition of the oxygen-evolving apparatus by alkaline intrathylakoid pH.     

Adenosine diphosphate moiety does not participate in structural changes for the signaling state in the sensor of blue-light using FAD domain of AppA

A sensor of blue light using FAD (BLUF) protein is a flavin adenine dinucleotide (FAD) based new class blue-light sensory flavoprotein. The BLUF domain of AppA was reconstituted in vitro from apoprotein and flavin adenine dinucleotide, flavin adenine mononucleotide or riboflavin. The light-induced FTIR spectra of the domain reconstituted from various flavins and the 13C-labeled apoprotein showed that identical light-induced structural changes occur in both the flavin chromophore and protein for the signaling state in all of the reconstituted holoproteins. The results showed that an adenosine 5'-dinucleotide moiety is not required for signaling-state formation in a BLUF domain.     

Determinants of Substrate Binding and Protonation in the Flavoenzyme Xenobiotic Reductase A

Xenobiotic reductase A (XenA) from Pseudomonas putida 86 catalyzes the NAD(P)H-dependent reduction of various α,β-unsaturated carbonyl compounds and is a member of the old yellow enzyme family. The reaction of XenA follows a ping-pong mechanism, implying that its active site has to accommodate and correctly position the various substrates to be oxidized (NADH/NADPH) and to be reduced (different α,β-unsaturated carbonyl compounds) to enable formal hydride transfers between the substrate and the isoalloxazine ring.The active site of XenA is lined by two tyrosine (Tyr27, Tyr183) and two tryptophan (Trp302, Trp358) residues, which were proposed to contribute to substrate binding. We analyzed the individual contributions of the four residues, using site-directed mutagenesis, steady-state and transient kinetics, redox potentiometry and crystal structure analysis. The Y183F substitution decreases the affinity of XenA for NADPH and reduces the rate of the oxidative half-reaction by two to three orders of magnitude, the latter being in agreement with its function as a proton donor in the oxidative half-reaction. Upon reduction of the flavin, Trp302 swings into the active site of XenA (in-conformation) and decreases the extent of the substrate-binding pocket. Its exchange against alanine induces substrate inhibition at elevated NADPH concentrations, indicating that the in-conformation of Trp302 helps to disfavor the nonproductive NADPH binding in the reduced state of XenA.Our analysis shows that while the principal catalytic mechanism of XenA, for example, type of proton donor, is analogous to that of other members of the old yellow enzyme family, its strategy to correctly position and accommodate different substrates is unprecedented.     

Aniline hydroxylase activities of haemoglobin: Kinetics and mechanism

The mechanism of the aniline hydroxylase activity of methaemoglobin in a monooxygenase system consisting of NADH as electron donor, riboflavin, FAD, FMN or methylene blue as electron carrier and methaemoglobin as the terminal oxidase has been studied. Hydrogen peroxide is produced from oxygen in a methaemoglobin-independent process. 4-Aminophenol is subsequently produced peroxidatively by an NADH-dependent process; NADH prevents a further oxidation of 4-aminophenol in the presence of haemoglobin. In the absence of electron carrier, NADH slowly reduces haemoglobin and then oxyhaemoglobin reacts with aniline to give 4-aminophenol. In the absence of electron donor and electron carrier, oxyhaemoglobin and aniline give rise to the reversible production of 4-aminophenol.     

Hydrogen bond switching among flavin and amino acid side chains in the BLUF photoreceptor observed by ultrafast infrared spectroscopy

BLUF domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae. BLUF domains are blue-light sensitive through a FAD cofactor that is involved in an extensive hydrogen-bond network with nearby amino acid side chains, including a highly conserved tyrosine and glutamine. The participation of particular amino acid side chains in the ultrafast hydrogen-bond switching reaction with FAD that underlies photoactivation of BLUF domains is assessed by means of ultrafast infrared spectroscopy. Blue-light absorption by FAD results in formation of FAD^•− and a bleach of the tyrosine ring vibrational mode on a picosecond timescale, showing that electron transfer from tyrosine to FAD constitutes the primary photochemistry. This interpretation is supported by the absence of a kinetic isotope effect on the fluorescence decay on H/D exchange. Subsequent protonation of FAD^•− to result in FADH^• on a picosecond timescale is evidenced by the appearance of a N-H bending mode at the FAD N5 protonation site and of a FADH^• C=N stretch marker mode, with tyrosine as the likely proton donor. FADH^• is reoxidized in 67 ps (180 ps in D₂O) to result in a long-lived hydrogen-bond switched network around FAD. This hydrogen-bond switch shows infrared signatures from the C-OH stretch of tyrosine and the FAD C4=O and C=N stretches, which indicate increased hydrogen-bond strength at all these sites. The results support a previously hypothesized rotation of glutamine by ∼180° through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch.     

Mechanism of foetal wastage following immunoneutralization of riboflavin carrier protein in the pregnant rat: disturbances in flavin coenzyme levels

Immunoneutralization of maternal RCP results in a greater than 90% decrease in the content and the incorporation of [2-14C]riboflavin into embryonic FAD as well as a percentage redistribution of both embryonic FMN and riboflavin. This is unaccompanied by any discernible changes in flavin distribution pattern in the maternal liver. Embryonic alpha-glycerophosphate dehydrogenase and NADPH-cytochrome c reductase register significant decreases in activities in the RCP antiserum-treated rats. These alterations readily explain the arrest of foetal growth culminating in pregnancy termination in the antiserum-treated animals.     

Addition of Signal Leader Sequences to the N-Termini of Olfactory Receptor Proteins Enhances Their Expression in Xenopus Oocyte

Several recent papers have reported the difficulties in expressing olfactory receptor proteins (ORs) in heterologous systems, and proposed that some sequences in ORs have negative effects on their efficient expression. To obtain an efficient expression system of ORs, we modified N-terminal sequences of ORs through the addition of exogenous sequences. Three kinds of sequences, designated as 5HT, V, and VL, were used. 5HT and V corresponded to the signal leader (SL) sequences of 5HT₃R and VIPR, respectively. VL corresponded to the first extracellular region of VIPR containing the SL sequence and three potential asparagine- (Asn-) linked glycosylation sites. The myc epitope was also added to the C-termini of the sequences. Several ORs including I7 of rat, GUST43 of rat, Y1 of medaka, FOR1-3 of pufferfish, 47E of carp, and ODR-10 of nematode were subjected to the modifications, and the RNAs encoding modified ORs were injected into Xenopus oocytes. The membrane fraction of the oocytes were analyszed by Western blotting to examine the expression of the proteins. In the cases of ORs modified with 5HT and V, only ODR-10 and 47E, both of which have more than two Asn-linked glycosylation sites in their extracellular regions, were detected as the bands of predicted molecular weights. On the other hand, most of the ORs modified with VL showed the bands of predicted molecular weights. These results suggest that SL sequences together with potential Asn-linked glycosylation sites have positive effects on the expression of ORs in heterologous systems.     

Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase

LadA, a long-chain alkane monooxygenase, utilizes a terminal oxidation pathway for the conversion of long-chain alkanes (up to at least C₃₆) to corresponding primary alcohols in thermophilic bacillus Geobacillus thermodenitrificans NG80-2. Here, we report the first structure of the long-chain alkane hydroxylase, LadA, and its complex with the flavin mononucleotide (FMN) coenzyme. LadA is characterized as a new member of the SsuD subfamily of the bacterial luciferase family via a surprising structural relationship. The LadA:FMN binary complex structure and a LadA:FMN:alkane model reveal a hydrophobic cavity that has dual roles: to provide a hydrogen-bond donor (His138) for catalysis and to create a solvent-free environment in which to stabilize the C4a-hydroperoxyflavin intermediate. Consequently, LadA should catalyze the conversion of long-chain alkanes via the acknowledged flavoprotein monooxygenase mechanism. This finding suggests that the ability of LadA to catalyze the degradation of long-chain alkanes is determined by the binding mode of the long-chain alkane substrates. The LadA structure opens a rational perspective to explore and alter the substrate binding site of LadA, with potential biotechnological applications in areas such as petroleum exploration and treatment of environmental oil pollution.     

Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level

Biochemical characterization of Corynebacterium ammoniagenes FADS (CaFADS) pointed to certain confusion about the stoichiometry of this bifunctional enzyme involved in the production of FMN and FAD in prokaryotes. Resolution of its crystal structure suggested that it might produce a hexameric ensemble formed by a dimer of trimers. We used atomic force microscopy (AFM) to direct imaging single CaFADS molecules bound to mica surfaces, while preserving their catalytic properties. AFM allowed solving individual CaFADS monomers, for which it was even possible to distinguish their sub-molecular individual N- and C-terminal modules in the elongated enzyme. Differences between monomers and higher stoichiometries were easily imaged, enabling us to detect formation of oligomeric species induced by ligand binding. The presence of ATP:Mg^2 + particularly induced the appearance of the hexameric assembly whose mean molecular volume resembles the crystallographic dimer of trimers. Finally, the AFM results are confirmed in cross-linking solution, and the presence of such oligomeric CaFADS species detected in cell extracts. All these results are consistent with the formation of a dimer of trimers during the enzyme catalytic cycle that might bear biological relevance.     

X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O₂ could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.     

Molecular cloning, characterisation and ligand-bound structure of an azoreductase from Pseudomonas aeruginosa

The gene PA0785 from Pseudomonas aeruginosa strain PAO1, which is annotated as a probable acyl carrier protein phosphodiesterase (acpD), has been cloned and heterologously overexpressed in Escherichia coli. The purified recombinant enzyme exhibits activity corresponding to that of azoreductase but not acpD. Each recombinant protein molecule has an estimated molecular mass of 23,050 Da and one non-covalently bound FMN as co-factor. This enzyme, now identified as azoreductase 1 from Pseudomonas aeruginosa (paAzoR1), is a flavodoxin-like protein with an apparent molecular mass of 110 kDa as determined by gel-filtration chromatography, indicating that the protein is likely to be tetrameric in solution. The three-dimensional structure of paAzoR1, in complex with the substrate methyl red, was solved at a resolution of 2.18 A by X-ray crystallography. The protein exists as a dimer of dimers in the crystal lattice, with two spatially separated active sites per dimer, and the active site of paAzoR1 was shown to be a well-conserved hydrophobic pocket formed between two monomers. The paAzoR1 enzyme is able to reduce different classes of azo dyes and activate several azo pro-drugs used in the treatment of inflammatory bowel disease (IBD). During azo reduction, FMN serves as a redox centre in the electron-transferring system by mediating the electron transfer from NAD(P)H to the azo substrate. The spectral properties of paAzoR1 demonstrate the hydrophobic interaction between FMN and the active site in the protein. The structure of the ligand-bound protein also highlights the pi-stacking interactions between FMN and the azo substrate.