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-Å 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° rotation of the DNA-binding domains that is best described as a jumping-jack-like motion.

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.

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.

Different from eukaryotes, the bacterial signal recognition particle (SRP) receptor lacks a membrane-tethering SRP receptor (SR) β subunit and is composed of only the SRα 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 SRα 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.

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.

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.

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.

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 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.

ABSTRACT 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.

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 SRα subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRβ 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.

The major source of surprise to us is the fact that none of the many groups who were/are working on the theme of nephritogenic antigens in acute poststreptococcal glomerulonephritis (APSGN) have published comparative studies. Our investigation was an attempt to remedy this situation. Before addressing the points raised by Yoshizawa et al., we wish to emphasize that there is now agreement on the data concerning streptococcal zymogen/proteinase (SPE B) in APSGN. Glomerular deposition of SPE B has been independently verified,1.Cu G.A. Mezzano S. Bannan J.D. Zabriskie J.B. Immunohistochemical and serological evidence for the role of streptococcal proteinase in acute post-streptococcal glomerulonephritis.Kidney Int. 1998; 54: 819-826Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholarand has been confirmed by Yoshizawa et al. in their APSGN biopsies using antibody supplied by us (personal communication). In addition, a number of independent studies have shown antibody to SPE B to be an excellent marker of group A streptococcus infection in general (see Batsford et al.2.Batsford S.R. Mezzano S. Mihatsch M. et al.Is the nephritogenic antigen in post-streptococcal glomerulonephritis pyrogenic exotoxin B (SPE B) or GAPDH?.Kidney Int. 2005; 68: 1120-1129Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and it is probably the best available serological marker for APSGN.3.Parra G. Rodriguez-Iturbe B. Batsford S. et al.Antibody to streptococcal zymogen in the serum of patients with acute glomerulonephritis: A multicentric study.Kidney Int. 1998; 54: 509-517Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar The second streptococcal component studied by us was glyceraldehyde-3-phosphate dehydrogenase (GAPDH), known to be identical to plasmin receptor from group A streptococcus4.Winram S.B. Lottenberg R. The plasmin-binding protein Plr of group A streptococci is identified as glyceraldehyde-3-phosphate dehydrogenase.Microbiology. 1996; 142: 2311-2320Crossref PubMed Scopus (144) Google Scholarand sharing at least 98% homology to nephritis-associated plasmin receptor, as reported by Yoshizawa et al.5.Yamakami K. Yoshizawa N. Wakabayashi K. et al.The potential role of for nephritis-associated plasmin receptor in acute post streptococcal glomerulonephritis.Methods. 2000; 21: 185-197Crossref PubMed Scopus (62) Google Scholarthemselves. We found glomerular deposition of GAPDH in 3/17 biopsies and antibody to GAPDH in 5/47 serum samples from APSGN patients. These results demonstrate that more than one antigen may be involved in the pathogenesis of APSGN. The lower frequency of positive staining for GAPDH, found by us, is not easily explained by methodology; indirect immunofluorescence is actually more sensitive than direct staining and has been successfully used to detect glomerular deposition of many different antigens, as shown in myriad previous reports. The indirect technique is more likely to produce false-positive rather than false-negative results. Minor differences in mobility of fractions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reported between laboratories is a common phenomena of a technical nature, it does not constitute proof of real differences between antigens. Yoshizawa et al. used a quantitative immunoblot method to assess serum antibody; this is not state of the art, it should be confirmed by enzyme-linked immunosorbent assay testing. If Yoshizawa et al. are convinced of the existence of isoforms of GAPDH/plasmin receptor, they should perform complete sequencing of their antigen preparation, this task is not trivial but with modern technology it no longer poses great problems.

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.

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.

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.

The aim of the present study was to identify released proteins of Streptococcus agalactiae and to investigate their immunoreactivity with human sera to determine whether such proteins might be viable as carrier proteins in conjugate vaccines. Infections with S. agalactiae are the leading cause of sepsis and meningitis in neonates. Vaccination of women of childbearing age would be a desirable alternative to intrapartum antibiotic prophylaxis, but factors that mediate S. agalactiae invasive disease and virulence are poorly defined. Capsule-based vaccines have shown only low immunogenicity to date, and interest has shifted towards S. agalactiae proteins, either as candidate vaccine antigens or as carrier proteins for serotype-specific S. agalactiae polysaccharides. In this study, some major released proteins of S. agalactiae could be identified, including molecules known to be present on the surface of bacterial cells but not previously described as released proteins, such as CAMP factor, a phosphocarrier protein, aldolase, enolase, PcsB, and heat-shock protein 70. Serotype-specific differences in the protein patterns of extracellular products and immunoreactivity with human sera could be detected by SDS-PAGE and Western blot. The identification of unexpected released proteins may indicate secondary functions for these proteins. In addition, the widespread immunoreactivity of these proteins with human sera as shown by Western blot indicates that released proteins may be promising candidates as carrier proteins in conjugate vaccines.

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.

The tryptic FAD-peptide carrying the flavin in 8alpha-(N3)histidyl linkage as natural hapten was isolated by HPLC from the bacterial enzyme 6-hydroxy-d-nicotine oxidase. The same flavin protein linkage is found in the mitochondrial succinate dehydrogenase flavoprotein subunit, the predominant flavoprotein with covalently bound FAD in mitochondria of cardiomyocytes. Peripheral blood mononuclear cells (PBMC) were isolated from four patients with acute myocarditis, seven patients with dilated cardiomyopathy (DCM) and from four healthy control individuals. The response of PBMC to the FAD-peptide was evaluated by measuring proliferation ([3H]-dThd incorporation) and cytokine secretion [interferon (IFN)-gamma]. PBMC from all patients with acute myocarditis showed positive responses to the FAD-peptide, in contrast to PBMC from patients with DCM or control individuals. Following the recovery of the patients from the acute inflammation of the heart, PBMC no longer exhibited a proliferation response to the FAD-peptide. A chemically synthesized FAD-free peptide with identical amino acid sequence induced no response of PBMC. The results are consistent with a recall response by activated T cells, specific for the normally cryptic mitochondrial flavin-hapten, which may be liberated following cardiomyocyte destruction during the inflammation of the heart.

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.