Insight into the induction mechanism of the GntR/HutC bacterial transcription regulator YvoA.

YvoA is a GntR/HutC transcription regulator from Bacillus subtilis implicated in the regulation of genes from the N-acetylglucosamine-degrading pathway. Its 2.4-A crystal structure reveals a homodimeric assembly with each monomer displaying a two-domain fold. The C-terminal domain, which binds the effector N-acetylglucosamine-6-phosphate, adopts a chorismate lyase fold, whereas the N-terminal domain contains a winged helix-turn-helix DNA-binding domain. Isothermal titration calorimetry and site-directed mutagenesis revealed that the effector-binding site in YvoA coincides with the active site of related chorismate lyase from Escherichia coli. The characterization of the DNA- and effector-binding properties of two disulfide-bridged mutants that lock YvoA in two distinct conformational states provides for the first time detailed insight into the allosteric mechanism through which effector binding modulates DNA binding and, thereby regulates transcription in a representative GntR/HutC family member. Central to this allosteric coupling mechanism is a loop-to-helix transition with the dipole of the newly formed helix pointing toward the phosphate of the effector. This transition goes in hand with the emergence of internal symmetry in the effector-binding domain and, in addition, leads to a 122 degrees rotation of the DNA-binding domains that is best described as a jumping-jack-like motion.

Twin-arginine-dependent translocation of SufI in the absence of cytosolic helper proteins.

The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Folding of some Tat precursor proteins requires dedicated chaperones that also sequester the signal sequence during the maturation process. Whether or not signal sequence-binding chaperones are a general prerequisite for all Tat substrate proteins is not known. Here, we have studied the propensity of Tat signal sequences of Escherichia coli to interact with general chaperones and peptidyl-prolyl-cis,trans-isomerases. Site-specific photocross-linking revealed a clear specificity for FK506-binding proteins. Nevertheless transport of the Tat substrate SufI into inverted inner membrane vesicles of E. coli was found to occur in the bona fide absence of any cytosolic chaperone. Our results suggest that in E. coli, cytosolic chaperones are not essential for the twin-arginine-dependent export of cofactor-less substrates.

Heterologous production, isolation, characterization and crystallization of a soluble fragment of the NADH:ubiquinone oxidoreductase (complex I) from Aquifex aeolicus.

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) is the first enzyme complex of the respiratory chains in many bacteria and most eukaryotes. It is the least understood of all, due to its enormous size and unique energy conversion mechanism. The bacterial complex is in general made up of 14 different subunits named NuoA-N. Subunits NuoE, -F, and -G comprise the electron input part of the complex. We have cloned these genes from the hyperthermophilic bacterium Aquifex aeolicus and expressed them heterologously in Escherichia coli. A soluble subcomplex made up of NuoE and NuoF and containing the NADH binding site, the primary electron acceptor flavin mononucleotide (FMN), the binuclear iron-sulfur cluster N1a, and the tetranuclear iron-sulfur cluster N3 was isolated by chromatographic methods. The proteins were identified by N-terminal sequencing and mass spectrometry; the cofactors were characterized by UV/vis and EPR spectroscopy. Subunit NuoG was not produced in this strain. The preparation was thermostable and exhibited maximum NADH/ferricyanide oxidoreductase activity at 85 degrees C. Analytical size-exclusion chromatography and dynamic light scattering revealed the homogeneity of the preparation. First attempts to crystallize the preparation led to crystals diffracting more than 2 A.

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR.

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder-order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates.

Expression and properties of arginyl-tRNA synthetase from jack bean (Canavalia ensiformis).

The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology. The thioredoxin component and the His-tag portion of the fused protein could be removed with thrombin, resulting in a homogeneous product retaining an N-terminal extension of 3.2 kDa compared to the native arginyl-tRNA synthetase. Both full-length fusion and thrombin-treated products proved to be active in aminoacylation, with similar kinetic parameters.

A cleavable N-terminal membrane anchor is involved in membrane binding of the Escherichia coli SRP receptor.

Different from eukaryotes, the bacterial signal recognition particle (SRP) receptor lacks a membrane-tethering SRP receptor (SR) beta subunit and is composed of only the SR alpha homologue FtsY. FtsY is a modular protein composed of three domains. The N- and G-domains of FtsY are highly similar to the corresponding domains of Ffh/SRP54 and SR alpha and constitute the essential core of FtsY. In contrast, the weakly conserved N-terminal A-domain does not seem to be essential, and its exact function is unknown. Our data show that a 14-amino-acid-long positively charged region at the N-terminus of the A-domain is involved in stabilizing the FtsY-SecYEG interaction. Mutant analyses reveal that the positively charged residues are crucial for this function, and we propose that the 14-amino-acid region serves as a transient lipid anchor. In its absence, the activity of FtsY to support cotranslational integration is reduced to about 50%. Strikingly, in vivo, a truncated isoform of FtsY that lacks exactly these first 14 amino acids exists. Different from full-length FtsY, which primarily cofractionates with the membrane, the N-terminally truncated isoform is primarily present in the soluble fraction. Mutating the conserved glycine residue at position 14 prevents the formation of the truncated isoform and impairs the activity of FtsY in cotranslational targeting. These data suggest that membrane binding and function of FtsY are in part regulated by proteolytic cleavage of the conserved 14-amino-acid motif.

PRMT6-mediated methylation of R2 in histone H3 antagonizes H3 K4 trimethylation.

The arginine methyltransferase PRMT6 (protein arginine methyltransferase 6) has been shown recently to regulate DNA repair and gene expression. As arginine methylation of histones is an important mechanism in transcriptional regulation, we asked whether PRMT6 possesses activity toward histones. We show here that PRMT6 methylates histone H3 at R2 and histones H4/H2A at R3 in vitro. Overexpression and knockdown analysis identify PRMT6 as the major H3 R2 methyltransferase in vivo. We find that H3 R2 methylation inhibits H3 K4 trimethylation and recruitment of WDR5, a subunit of the MLL (mixed lineage leukemia) K4 methyltransferase complex, to histone H3 in vitro. Upon PRMT6 overexpression, transcription of Hox genes and Myc-dependent genes, both well-known targets of H3 K4 trimethylation, decreases. This transcriptional repression coincides with enhanced occurrence of H3 R2 methylation and PRMT6 as well as reduced levels of H3 K4 trimethylation and MLL1/WDR5 recruitment at the HoxA2 gene. Upon retinoic acid-induced transcriptional activation of HoxA2 in a cell model of neuronal differentiation, PRMT6 recruitment and H3 R2 methylation are diminished and H3 K4 trimethylation increases at the gene. Our findings identify PRMT6 as the mammalian methyltransferase for H3 R2 and establish the enzyme as a crucial negative regulator of H3 K4 trimethylation and transcriptional activation.

The toposome, essential for sea urchin cell adhesion and development, is a modified iron-less calcium-binding transferrin.

We describe the structure and function of the toposome, a modified calcium-binding, iron-less transferrin, the first member of a new class of cell adhesion proteins. In addition to the amino acid sequence of the precursor, we determined by Edman degradation the N-terminal amino acid sequences of the mature hexameric glycoprotein present in the egg as well as that of its derived proteolytically modified fragments necessary for development beyond the blastula stage. The approximate C-termini of the fragments were determined by a combination of mass spectrometry and migration in reducing gels before and after deglycosylation. This new member of the transferrin family shows special features which explain its evolutionary adaptation to development and adhesive function in sea urchin embryos: (i) a protease-inhibiting WAP domain, (ii) a 280 amino acid cysteine-less insertion in the C-terminal lobe, and (iii) a 240 residue C-terminal extension with a modified cystine knot motif found in multisubunit external cell surface glycoproteins. Proteolytic removal of the N-terminal WAP domain generates the mature toposome present in the oocyte. The modified cystine knot motif stabilizes cell-bound trimers upon Ca-dependent dissociation of hexamer-linked cells. We determined the positions of the developmentally regulated cuts in the cysteine-less insertion, which produce the fragments observed previously. These fragments remain bound to the hexameric 22S particle in vivo and are released only after treatment of the purified toposome with reducing agents. In addition, some soluble smaller fragments with possible signal function are produced. Sequence comparison of five sea urchin species reveals the location of the cell-cell contact site targeted by the species-specific embryo dissociating antibodies. The evolutionary tree of 2-, 1-, and 0-ferric transferrins implies their evolution from a basic cation-activated allosteric design modified to serve multiple functions.

A derivative of lipid A is involved in signal recognition particle/SecYEG-dependent and -independent membrane integrations.

A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by signal recognition particle and signal recognition particle receptor. Integration of M13 procoat, which is independent of both signal recognition particle/signal recognition particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.

Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites.

Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRbeta subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane. However, the mechanisms that are involved in this membrane association are only poorly understood. Our data indicate that membrane association of FtsY involves two distinct binding sites and that binding to both sites is stabilized by blocking its GTPase activity. Binding to the first site requires only the NG-domain of FtsY and confers protease protection to FtsY. Importantly, the SecY translocon provides the second binding site, to which FtsY binds to form a carbonate-resistant 400-kD FtsY-SecY translocon complex. This interaction is stabilized by the N-terminal A-domain of FtsY, which probably serves as a transient lipid anchor.

A functional mobA gene for molybdopterin cytosine dinucleotide cofactor biosynthesis is required for activity and holoenzyme assembly of the heterotrimeric nicotine dehydrogenases of Arthrobacter nicotinovorans.

Two Arthrobacter nicotinovorans molybdenum enzymes hydroxylate the pyridine ring of nicotine. Molybdopterin cytosine dinucleotide (MCD) was determined to be a cofactor of these enzymes. A mobA gene responsible for the formation of MCD could be identified and its function shown to be required for assembly of the heterotrimeric molybdenum enzymes.

An alpha/beta-fold C--C bond hydrolase is involved in a central step of nicotine catabolism by Arthrobacter nicotinovorans.

The enzyme catalyzing the hydrolytic cleavage of 2,6-dihydroxypseudooxynicotine to 2,6-dihydroxypyridine and gamma-N-methylaminobutyrate was found to be encoded on pAO1 of Arthrobacter nicotinovorans. The new enzyme answers an old question about nicotine catabolism and may be the first C--C bond hydrolase that is involved in the biodegradation of a heterocyclic compound.

Is the nephritogenic antigen in post-streptococcal glomerulonephritis pyrogenic exotoxin B (SPE B) or GAPDH?

BACKGROUND: Acute glomerulonephritis can follow infection by group A streptococci. An immune-complex pathogenesis is accepted, but the causative antigen(s) is still controversial. In recent years, 2 streptococcal antigens, the cationic cysteine proteinase exotoxin B (SPE B) and the plasmin receptor, a glyceraldehyde phosphate dehydrogenase (Plr, GAPDH) have attracted attention because: (1) they were localized in glomeruli in patients with acute post-streptococcal glomerulonephritis (APSGN); and (2) serum antibody to these antigens was associated with nephritogenic streptococcal infections. To date, putative nephritogens were always tested independently. Here, the relevance of SPE B and GAPDH was evaluated in the same renal biopsies and serum samples of well-defined APSGN patients. METHODS: Renal biopsies (17 patients) and serum samples (53 patients) with APSGN and appropriate controls were examined. Immunofluorescent staining of frozen sections was performed using specific antibodies to SPE B and GAPDH. Serum antibodies were investigated by both enzyme-linked immunosorbent assay (ELISA) and Western blot methodology. RESULTS: Glomerular deposits of SPE B were demonstrated in 12/17 APSGN biopsies, and 2 cases were borderline; circulating antibodies were found in all instances (53/53 patients). Glomerular deposition of GAPDH was detected in 1/17 biopsies, and 2 cases were borderline; circulating antibodies were found in 5/47 patients. In 31 control biopsies, only weak staining for each antigen was found in 2 cases. CONCLUSION: In this study, glomerular deposits of and antibody response to zymogen/SPE B are more consistently present in APSGN than deposits and antibody response to GAPDH. Zymogen/SPE B is likely to be the major antigen involved in the pathogenesis of most cases of APSGN.

A novel gamma-N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1.

Nicotine catabolism, linked in Arthrobacter nicotinovorans to the presence of the megaplasmid pAO1, leads to the formation of gamma-N-methylaminobutyrate from the pyrrolidine ring of the alkaloid. Until now the metabolic fate of gamma-N-methylaminobutyrate has been unknown. pAO1 carries a cluster of ORFs with similarity to sarcosine and dimethylglycine dehydrogenases and oxidases, to the bifunctional enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase and to formyltetrahydrofolate deformylase. We cloned and expressed the gene carrying the sarcosine dehydrogenase-like ORF and showed, by enzyme activity, spectrophotometric methods and identification of the reaction product as gamma-aminobutyrate, that the predicted 89 395 Da flavoprotein is a demethylating gamma-N-methylaminobutyrate oxidase. Site-directed mutagenesis identified His67 as the site of covalent attachment of FAD and confirmed Trp66 as essential for FAD binding, for enzyme activity and for the spectral properties of the wild-type enzyme. A Km of 140 microm and a kcat of 800 s(-1) was determined when gamma-N-methylaminobutyrate was used as the substrate. Sarcosine was also turned over by the enzyme, but at a rate 200-fold slower than gamma-N-methylaminobutyrate. This novel enzyme activity revealed that the first step in channelling the gamma-N-methylaminobutyrate generated from nicotine into the cell metabolism proceeds by its oxidative demethylation.

Composition and activity of the Rhodobacter capsulatus degradosome vary under different oxygen concentrations.

The influence of changes in temperature or oxygen tension during growth of Rhodobacter capsulatus on the composition and activity of the degradosome, an RNA-processing protein complex, was investigated. Only minor differences in the amount of specific proteins of the complex were observed after a decrease or increase of the temperature, but dramatic variations were detectable during growth at different oxygen concentrations. In particular, the amount of the transcription factor Rho, which was previously shown to be associated with the R. capsulatus degradosome, was strongly increased under aerobic conditions. Remarkably, oxygen tension oppositely affected the levels of the two helicases associated with the degradosome. RNase E and the degradosome from aerobically grown cultures degraded a transcript which represents part of the puf operon encoding proteins of the photosynthetic apparatus faster than did the degradosome from semiaerobically grown cultures.

Identification of beta-subunit of bacterial RNA-polymerase--a non-species-specific bacterial protein--as target of antibodies in primary biliary cirrhosis.

Several observations suggest that bacteria induce autoimmunity in primary biliary cirrhosis (PBC). Since no PBC-specific bacterial species could be identified, it can be speculated that the triggers are non-species-specific bacterial proteins. This hypothesis would imply that several or even all bacterial species can trigger PBC. Therefore, we investigated whether PBC exhibits immune reactions to non-species-specific bacterial antigens. Yersinia enterocolitica O3 was screened for the presence of proteins that were labeled by immunoblotting using PBC sera. We focused our investigations on a 160-kDa protein, which was further enriched and characterized by partial N-terminal amino acid sequencing. The prevalence of antibodies to this protein was determined by immunoblotting in a variety of diseases. The 160-kDa protein was identified as the beta-subunit of bacterial RNA-polymerase, a highly conserved bacterial protein with a very high degree of sequence identity among all bacterial species. Antibodies to the beta-subunit of bacterial RNA polymerase were specific for this protein. Until now no mammalian protein could be found that cross-reacts with these antibodies. The prevalence of antibodies to the beta-subunit of bacterial RNA polymerase (ARPA) using the protein from Yersinia enterocolitica O3 (serum dilution 1:1000) was: healthy controls (HC, N = 101) 7.9%, primary biliary cirrhosis (PBC, N = 61) 32.8%, autoimmune hepatitis type 1 (AIH, N = 46) 26.1%, alcoholic liver cirrhosis (ALC, N = 44) 9.1%, Crohn's disease (CD, N = 38) 7.9%, ulcerative colitis (UC, N = 24) 8.3%, primary sclerosing cholangitis + UC (PSC/UC, N = 11) 0%, acute yersiniosis (Yers, N = 36) 19.4%, acute infection with Campylobacter jejuni (Camp, N = 10) 0%, acute Q-fever (QF, N = 16) 6.25%, chronic hepatitis C (HCV, N = 39) 7.7%, c-ANCA-positive vasculitis (Vasc, N = 40) 15%, systemic lupus erythematosus (SLE, N = 28) 10.7%, and malaria tropica (MT, N = 24) 16.7%. There was no significant difference between PBC and AIH. The group of autoimmune liver diseases (PBC + AIH, N = 107, 29.9%) differed highly significantly from HC, chronic inflammatory bowel diseases (CD + UC + PSC/UC, N = 73, 6.8%), ALC, and HCV and also differed significantly (P = 0.01) from the group with bacterial and parasitic diseases (Yers + Camp + QF + MT, N = 86,13.95%) and from the group with Vasc + SLE (N = 68,13.2%). Testing of ARPA using the protein from E. coli yielded nearly identical results. In conclusion, an increased prevalence of antibodies to the beta-subunit of bacterial RNA polymerase, a highly conserved non-species-specific bacterial protein, can be found in primary biliary cirrhosis, but also in autoimmune hepatitis type I. These findings do not add an argument for a bacterial trigger of PBC. Rather, they suggest that ARPA belong to the pool of natural antibodies that are up-regulated in autoimmune liver diseases.

Two W-containing formate dehydrogenases (CO2-reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidans.

Two formate dehydrogenases (CO2-reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO2-reduction rates, and low Km values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 U.mg-1 and a Km for formate of 0.04 mm were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 U.mg-1 and a Km of 0.01 mm for formate with benzyl viologen as electron acceptor. The specific CO2-reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 U.mg-1 and 89 U.mg-1, respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 +/- 3, 56 +/- 2 and 19 +/- 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 +/- 3 kDa and a 33 +/- 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected. EPR spectroscopy suggested that FDH-1 contains at least four [2Fe-2S] clusters per molecule and additionally paramagnetically coupled [4Fe-4S] clusters. FDH-2 contains at least two [4Fe-4S] clusters per molecule. As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters.

Characterization of the chlorate reductase from Pseudomonas chloritidismutans.

A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate. Differences were also found in N-terminal sequences, molecular weight, and subunit composition. Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are also found in other dissimilatory oxyanion reductases.

Sequence analysis reveals new membrane anchor of reaction centre-bound cytochromes possibly related to PufX.

Most of the bacterial photosynthetic reaction centres known to date contain a cytochrome subunit with four covalently bound haem groups. In the case of Blastochloris viridis, this reaction centre subunit is anchored in the membrane by a lipid molecule covalently attached to the cysteine which forms the N-terminus of the mature protein after processing by a signal peptidase. We show that posttranslational N-terminal cleavage of the cytochrome subunit does not occur in the aerobic photosynthetic bacterium Roseobacter denitrificans. From sequence analysis of the resulting elongated N-terminus it follows that a transmembrane helix is anchoring the reaction centre-bound cytochrome in the membrane. Comparative sequence analysis strongly suggests that all cytochrome subunits lacking the lipid coupling cysteine share this structural feature. Comparison of the N-terminal segment of the cytochrome subunit of Roseobacter denitrificans with the sequences of the PufX proteins from Rhodobacter sphaeroides and Rhodobacter capsulatus suggests a phylogenetic relation.

Hemin binding, functional expression, and complementation analysis of Pap 31 from Bartonella henselae.

Growth of Bartonella henselae is strongly heme dependent, and B. henselae is unable to synthesize heme itself. At least five outer membrane-associated proteins from B. henselae bind hemin, including the 31-kDa protein designated Pap31. The gene of this protein was heterologously expressed in Escherichia coli M15(pREP4) and detected with monoclonal antibodies in the outer membrane fraction. Complementation of the hemA-deficient mutant E. coli K-12 EB53 (aroB tsx malT hemA) with pap31 demonstrated that this protein is involved in heme acquisition and may be an important virulence factor in the pathogenesis of B. henselae.