Mechanism of action of S-ribosylhomocysteinase (LuxS)

S-Ribosylhomocysteinase (LuxS) cleaves the thioether bond in S-ribosylhomocysteine to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione. This reaction serves the dual purposes of detoxification of S-adenosylhomocysteine and production of type 2 quorum sensing molecule. Recent research has shown that LuxS uses Fe(2+) to catalyze an internal redox reaction, shifting the ribose carbonyl group from its C1 to C3 position. Subsequent beta-elimination completes this highly unusual reaction. LuxS and other enzymes on the same pathway may provide a novel class of antibacterial drug targets.     

Signaling system in Porphyromonas gingivalis based on a LuxS protein

The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.     

Nuclear coded mitochondrial protein prohibitin is an iron regulated iron binding protein

Mitochondria are central to iron homeostasis. However, various proteins involved in iron metabolism inside the mitochondria are still to be identified. Herein we report that nuclear coded mitochondrial protein prohibitin binds to iron and involved in intracellular iron homeostasis. Like other iron regulated proteins, prohibitin mRNA contains functional iron-response element and is regulated by intracellular iron levels. Tyrosine residues involved in iron binding attribute of prohibitin are identified using site-directed mutagenesis. These data together suggest that prohibitin functions as an intracellular iron binding protein and plays a role in intracellular iron homeostasis.     

FAST is a BCL-X(L)-associated mitochondrial protein

The TIA-1-interacting protein Fas-activated serine/threonine phosphoprotein (FAST) is a component of a signaling cascade that is initiated by ligation of the Fas receptor. Immunofluorescence microscopy using affinity-purified antibodies raised against recombinant FAST reveals that the endogenous protein associates with mitochondria. Subcellular fractionation confirms that FAST is a component of mitochondria. FAST is tethered to mitochondria by a lysine/arginine-rich domain at its carboxyl terminus that is structurally similar to the mitochondrial tethering motifs of monoamine oxidase B and cytochrome b5. At the mitochondrial membrane, FAST interacts with BCL-X(L). The BCL-X(L) binding domain maps to a BCL-2-homology-3 (BH3)-related domain that is distinct from the mitochondrial-tethering domain (MTD). Although interactions between FAST and BCL-X(L) require both the BH3-related domain and the MTD, the requirement for mitochondrial tethering can be conferred by a heterologous MTD. Our results suggest that FAST-BCL-X(L) interactions are likely to regulate mitochondrial metabolism during Fas-induced apoptosis.     

Crystal structure of S-ribosylhomocysteinase (LuxS) in complex with a catalytic 2-ketone intermediate

S-Ribosylhomocysteinase (LuxS) is an Fe(2+)-dependent metalloenzyme that catalyzes the cleavage of the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of type II bacterial quorum-sensing molecule. The proposed mechanism involves an initial metal-catalyzed aldose-ketose isomerization reaction, which results in the migration of the ribose carbonyl group from its C1 to C2 position and the formation of a 2-ketone intermediate. A repetition of the isomerization reaction shifts the carbonyl group to the C3 position. Subsequent beta-elimination reaction at the C4 and C5 positions completes the catalytic cycle. In this work, a catalytically inactive mutant (C84A) of Co(2+)-substituted Bacillus subtilis LuxS was cocrystallized with the 2-ketone intermediate and the structure was determined to 1.8 A resolution. The structure reveals that the C2 carbonyl oxygen is directly coordinated with the metal ion, providing strong support for the proposed Lewis acid function of the metal ion during catalysis. Cys-84 and Glu-57 are optimally positioned to act as general acids/bases during the isomerization and elimination reactions. In addition, Ser-6, His-11, and Arg-39 are involved in substrate/ intermediate binding through hydrogen bonding interactions. The above conclusions are further confirmed by site-directed mutagenesis and visible absorption spectroscopic studies.     

Visinin-like protein (VILIP) is a neuron-specific calcium-dependent double-stranded RNA-binding protein.

Double-stranded RNA-binding proteins function in regulating the stability, translation, and localization of specific mRNAs. In this study, we have demonstrated that the neuron-specific, calcium-binding protein, visinin-like protein (VILIP) contains one double-stranded RNA-binding domain, a protein motif conserved among many double-stranded RNA-binding proteins. We showed that VILIP can specifically bind double-stranded RNA, and this interaction specifically requires the presence of calcium. Mobility shift studies indicated that VILIP binds double-stranded RNA as a single protein-RNA complex with an apparent equilibrium dissociation constant of 9.0 x 10(-6) M. To our knowledge, VILIP is the first double-stranded RNA-binding protein shown to be calcium-dependent. Furthermore, VILIP specifically binds the 3'-untranslated region of the neurotrophin receptor, trkB, an mRNA localized to hippocampal dendrites in an activity-dependent manner. Given that VILIP is also expressed in the hippocampus, these data suggest that VILIP may employ a novel, calcium-dependent mechanism to regulate its binding to important localized mRNAs in the central nervous system.     

TRAF-interacting protein (TRIP) is a RING-dependent ubiquitin ligase

TRAF-interacting protein (TRIP) was initially identified as a TRAF1- and TRAF2-binding partner that inhibited NF-kappaB activation without a known mechanism. Inspection of the TRIP sequence revealed an N-terminal RING domain, which is found in many E3 ubiquitin (Ub) ligases. We show that TRIP is a RING-dependent Ub ligase that undergoes auto-ubiquitination and requires an intact RING domain. Both TRIP and its RING mutant interact with TRAF1, 2, 3, 5, and 6, but failed to interact with CYLD and NIK. Stable expression of TRIP or a RING mutant did not affect IKK activation induced by TNF or IL-1 and had no affect on TNF-induced apoptosis. Similarly, RANKL-induced signaling and osteoclastogenesis were not affected by TRIP or its RING mutant. Interestingly, TRIP expression was down regulated during the late stages of osteoclastogenesis. Taken together, our results demonstrate that TRIP is a novel RING-dependent Ub ligase and a binding partner for TRAFs.     

Phorbol ester-induced mononuclear cell differentiation is blocked by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059.

The purpose of this study was to evaluate whether the mitogen-activated protein kinase (MAPK) signaling pathway contributes to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mononuclear differentiation in the human myeloblastic leukemia ML-1 cells. Upon TPA treatment, the activity of ERK1 and ERK2 rapidly increased, with maximal induction between 1 and 3 h, while ERK2 protein levels remained constant. The activity of JNK1 was also significantly induced, with JNK1 protein levels increasing moderately during exposure to TPA. Treatment of cells with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK), inhibited TPA-induced ERK2 activity. Furthermore, PD98059 completely blocked the TPA-induced differentiation of ML-1 cells, as assessed by a number of features associated with mononuclear differentiation including changes in morphology, nonspecific esterase activity, phagocytic ability, NADPH oxidase activity, mitochondrial respiration, and c-jun mRNA inducibility. We conclude that activation of the MEK/ERK signaling pathway is necessary for TPA-induced mononuclear cell differentiation.     

C-CAM (Cell-CAM 105) is a calmodulin binding protein.

C-CAM (Cell-CAM 105) is a transmembrane cell adhesion molecule belonging to the immunoglobulin superfamily. It mediates intercellular adhesion of rat hepatocytes and occurs in various isoforms in several epithelia, vessel endothelia and leukocytes. We now report that purified liver C-CAM interacts specifically with calmodulin. Binding was observed both when 125I-labeled C-CAM was used in a dot-blot assay and when 125I-labeled calmodulin was used in a gel overlay assay. Experiments with protease-generated peptides indicated that calmodulin bound to the cytoplasmic domain of C-CAM. Analyses of whole liver membranes demonstrated that C-CAM is one of five major proteins that bind calmodulin in a calcium-dependent manner.     

Human plasma retinol-binding protein (RBP4) is also a fatty acid-binding protein

RBP4 (plasma retinol-binding protein) is the 21?kDa transporter of all-trans retinol that circulates in plasma as a moderately tight 1:1 molar complex of the vitamin with the protein. RBP4 is primarily synthesized in the liver but is also produced by adipose tissue and circulates bound to a larger protein, transthyretin, TTR, that serves to increase its molecular mass and thus avoid its elimination by glomerular filtration.This paper reports the high resolution three-dimensional structures of human RBP4 naturally lacking bound retinol purified from plasma, urine and amniotic fluid. In all these crystals we found a fatty acid molecule bound in the hydrophobic ligand-binding site, a result confirmed by mass spectrometry measurements.In addition we also report the 1.5?Å resolution structures of human holo-RBP4 and of the protein saturated with palmitic and lauric acid and discuss the interaction of the fatty acids and retinol with the protein.     

The intracellular iron sensor calcein is catalytically oxidatively degraded by iron(II) in a hydrogen peroxide-dependent reaction

The fluorescent metal chelating dye calcein is used to obtain an estimate of cellular iron levels and to measure the kinetics of the entry of chelators and chelating drugs into cells. Under reducing conditions in the presence of ascorbic acid, such as that would be present in the cell, the Fe(II)-calcein complex was rapidly formed with a rate constant of 3 x 10(5) M(-1) s(-1). A slower iron-dependent catalytic degradation of calcein also occurred that resulted in the formation of a non-fluorescent calcein product. The Fe(II)-catalyzed degradation of calcein was largely, but not completely, prevented by catalase. Electron paramagnetic resonance spin trapping experiments showed that the Fe(II)-calcein complex promoted formation of hydroxyl or a hydroxyl radical-like species. Together these results indicated that Fe(II) catalyzed the degradation of calcein through both hydrogen peroxide, and to a lesser extent, non-hydrogen peroxide-dependent pathways. The iron-calcein complexes that were responsible for the degradation of calcein were likely high valence oxidizing iron-oxo species such as perferryl or ferryl complexes that were redox cycled by ascorbic acid. Thus, the use of calcein as an intracellular iron-sensing indicator may yield misleading results due to its degradation under certain conditions.     

The iron-sulfur cluster-free hydrogenase (Hmd) is a metalloenzyme with a novel iron binding motif

The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing cofactor of yet unknown structure. X-ray absorption spectroscopy of the active, as isolated enzyme from Methanothermobacter marburgensis (mHmd) and of the active, reconstituted enzyme from Methanocaldococcus jannaschii (jHmd) revealed the presence of mononuclear iron with two CO, one sulfur and one or two N/O in coordination distance. In jHmd, the single sulfur ligand is most probably provided by Cys176, as deduced from a comparison of the activity and of the x-ray absorption and M?ssbauer spectra of the enzyme mutated in any of the three conserved cysteines. In the isolated Hmd cofactor, two CO, one sulfur, and two nitrogen/oxygen atoms coordinate the iron, the sulfur ligand being most probably provided by mercaptoethanol, which is absolutely required for the extraction of the iron-containing cofactor from the holoenzyme and for the stabilization of the extracted cofactor. In active mHmd holoenzyme, the number of iron ligands increased by one when one of the Hmd inhibitors (CO or KCN) were present, indicating that in active Hmd, the iron contains an open coordination site, which is proposed to be the site of H2 interaction.     

Biochemical and spectroscopic studies on (S)-2-hydroxypropylphosphonic acid epoxidase: a novel mononuclear non-heme iron enzyme

The last step of the biosynthesis of fosfomycin, a clinically useful antibiotic, is the conversion of (S)-2-hydroxypropylphosphonic acid (HPP) to fosfomycin. Since the ring oxygen in fosfomycin has been shown in earlier feeding experiments to be derived from the hydroxyl group of HPP, this oxirane formation reaction is effectively a dehydrogenation process. To study this unique C-O bond formation step, we have overexpressed and purified the desired HPP epoxidase. Results reported herein provided initial biochemical evidence revealing that HPP epoxidase is an iron-dependent enzyme and that both NAD(P)H and a flavin or flavoprotein reductase are required for its activity. The 2 K EPR spectrum of oxidized iron-reconstituted fosfomycin epoxidase reveals resonances typical of S = (5)/(2) Fe(III) centers in at least two environments. Addition of HPP causes a redistribution with the appearance of at least two additional species, showing that the iron environment is perturbed. Exposure of this sample to NO elicits no changes, showing that the iron is nearly all in the Fe(III) state. However, addition of NO to the Fe(II) reconstituted enzyme that has not been exposed to O(2) yields an intense EPR spectrum typical of an S = (3)/(2) Fe(II)-NO complex. This complex is also heterogeneous, but addition of substrate converts it to a single, homogeneous S = (3)/(2) species with a new EPR spectrum, suggesting that substrate binds to or near the iron, thereby organizing the center. The fact that NO binds to the ferrous center suggests O(2) can also bind at this site as part of the catalytic cycle. Using purified epoxidase and (18)O isotopic labeled HPP, the retention of the hydroxyl oxygen of HPP in fosfomycin was demonstrated. While ether ring formation as a result of dehydrogenation of a secondary alcohol has precedence in the literature, these catalyses require alpha-ketoglutarate for activity. In contrast, HPP epoxidase is alpha-ketoglutarate independent. Thus, the cyclization of HPP to fosfomycin clearly represents an intriguing conversion beyond the scope entailed by common biological epoxidation and C-O bond formation.     

A rubredoxin-like mononuclear FeS4 derivative of adrenal iron-sulfur protein (adrenodoxin)

A rubredoxin-like mononuclear iron-sulfur derivative of adrenodoxin was prepared from the apoprotein and FeCl₃ in the presence of dithiothreitol. The mononuclear compound displayed optical absorption maxima at 276, 350, and 500 nm, and exhibited electron paramagnetic resonance absorption at g = 4.27 with a shoulder at g = 4.28, which can be ascribed to high spin ferric ion. From p-chloromercuriphenyl sulfonate titration experiments the iron atom appears to contain approximately one g atom of iron per mole of protein. This rubredoxin-like derivative was very unstable at 22° (the half-life was approximately 10 minutes), whereas the native 2 Fe2S1 protein is known to be quite stable. This instability is believed to be intrinsic to the polypeptide sequence of adrenodoxin.     

The barley 60 kDa jasmonate-induced protein (JIP60) is a novel ribosome-inactivating protein

The N-terminal region of a 60 kDa, jasmonate-induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosome-inactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N-terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticulocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome-inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.     

Fe(III) Oxide Reduction by Anaerobic Biofilm Formation-DeficientS-Ribosylhomocysteine Lyase (LuxS) Mutant of Shewanella oneidensis

Shewanella oneidensis respires a variety of terminal electron acceptors, including solid phase Fe(III) oxides. S. oneidensis transfers electrons to Fe(III) oxides via direct (outer membrane- or nanowire-localized c-type cytochromes) and indirect (electron shuttling and Fe(III) solubilization) pathways. In the present study, the influence of anaerobic biofilm formation on Fe(III) oxide reduction by S. oneidensis was determined. The gene encoding the activated methyl cycle (AMC) enzyme S-ribosylhomocysteine lyase (LuxS) was deleted in-frame to generate the corresponding mutant ΔluxS. Conventional biofilm assays and visual inspection via confocal laser scanning microscopy indicated that the wild-type strain formed anaerobic biofilms on Fe(III) oxide-coated silica surfaces, while the ΔluxS mutant was severely impaired in anaerobic biofilm formation on such surfaces. Cell-hematite attachment isotherms demonstrated that the ΔluxS mutant was also severely impaired in attachment to hematite surfaces under anaerobic conditions. The S. oneidensis ΔluxS mutant, however, reduced Fe(III) at wild-type rates during anaerobic incubation with Fe(III) oxide-coated silica surfaces or in batch cultures with Fe(III) oxide or hematite as a terminal electron acceptor. Anaerobic biofilm formation by the ΔluxS mutant was restored to wild-type rates by providing a wild-type copy of luxS in trans or by the addition of AMC or transsulfurylation pathway metabolites involved in organic sulfur metabolism. LuxS is thus required for wild-type anaerobic biofilm formation on Fe(III) oxide surfaces, yet the inability to form wild-type anaerobic biofilms on Fe(III) oxide surfaces does not alter Fe(III) oxide reduction activity.     

Uromodulin (Tamm-Horsfall glycoprotein/uromucoid) is a phosphatidylinositol-linked membrane protein

Uromodulin, originally identified as an immunosuppressive glycoprotein in the urine of pregnant women, has been previously shown to be identical to human Tamm-Horsfall glycoprotein (THP). THP is synthesized by the kidney and localizes to the renal thick ascending limb and early distal tubule. It is released into the urine in large quantities and thus represents a potential candidate for a protein secreted in a polarized fashion from the apical plasma membrane of epithelial cells in vivo. After introduction of the full-length cDNA encoding uromodulin/THP into HeLa, Caco-2, and Madin-Darby canine kidney cells by transfection, however, the expressed glycoprotein was almost exclusively cell-associated, as determined by immunoprecipitation after radioactive labeling of the cells. By immunofluorescence, THP was localized to the plasma membranes of transfected cells. In transfected cell extracts, THP also remained primarily in the detergent phase in a Triton X-114 partitioning assay, indicating that it has a hydrophobic character, in contrast to its behavior after isolation from human urine. Triton X-114 detergent-associated THP was redistributed to the aqueous phase after treatment of cell extracts with phosphatidylinositol-specific phospholipase C. Treatment of intact transfected HeLa cells with phosphatidylinositol-specific phospholipase C also resulted in the release of THP into the medium, suggesting that it is a glycosylphosphatidylinositol (GPI)-linked membrane protein. Similar to other known GPI-linked proteins, uromodulin/THP contains a stretch of 16 hydrophobic amino acids at its extreme carboxyl terminus which could function as a GPI addition signal and was shown to label with [3H]ethanolamine. The results indicate that THP is a member of this class of lipid-linked membrane proteins and is released into the urine after the loss of its hydrophobic anchor, probably by the action of a phospholipase or protease.