Emile Schiltz

The structure of porin from Rhodobacter capsulatus at 1.8 Å resolution

The structure of the porin from Rhodobacter capsulanus was determined at a resolution of 1.8 Å. The analysis started from a closely related crystal structure that had been solved at a medium resolution of 3 Å using multiple isomorphous replacement and solvent flattening. The new structure contains the complete sequence of 301 amino acid residues. Refinement of the model is under way: the present R‐factor is 22% with good geometry. Except for the lengths of several loops, the resulting chain fold corresponds to the medium resolution model. The membrane channel is lined by a large number of ionogenic side chains with characteristic segregation of differently charged groups.

Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism

Toluene is anoxically degraded to CO2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99‐), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox‐active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (α), 90 (α′), 12 (β) and 10 kDa (γ) were detected on sodium dodecyl sulphate (SDS) gels. The N‐terminal sequences of the α‐ and α′‐subunits were identical, suggesting a C‐terminal degradation of half of the α‐subunits to give the α′‐subunit. The composition of native enzyme therefore appears to be α2β2γ2. A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical‐containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the α′‐subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7‐promotor/polymerase system.

Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L.

Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.), and the product N-benzoylanthranilate is the precursor of several sets of dianthramides. The transferase activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract. The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa. Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of these sequences differed in a few amino acid residues only suggesting the presence of isoenzymes. A specific 0.8 kb cDNA probe was generated by RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)+ RNA from elicited carnation cells. Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1–3, were sequenced and shown to encode full-size N-benzoyltransferases. The translated peptide sequences revealed more than 95% identity among these three clones. The additional two clones harbored insert sequences mostly homologous with pchcbt1 but differing in the 3′-flanking regions due to variable usage of poly(A) addition sites. The identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequences as well as by the activity of the benzoyltransferase expressed in Escherichia coli. Therefore, carnation encodes a small family of anthranilate N-benzoyltransferase genes. In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Thus the cDNAs described represent also the first hydroxycinnamoyltransferases cloned from plants, which classifies the enzymes as hydroxycinnamoyl/benzoyltransferases.

Regulation of Ferritin-Mediated Cytoplasmic Iron Storage by the Ferric Uptake Regulator Homolog (Fur) of Helicobacter pylori

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.

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 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.

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.

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.

Prediction of the general structure of OmpF and PhoE from the sequence and structure of porin from Rhodobacter capsulatus. Orientation of porin in the membrane

By comparing the hydrophilicity profiles and sequences of porin from Rhodobacter capsulatus with those of OmpF and PhoE from Escherichia coli, a set of insertions and deletions for alignment of the sequences has been deduced. With this alignment a similar folding of OmpF and PhoE has been predicted as found by X-ray structure analysis of porin from Rhodobacter capsulates. Furthermore, the orientation of the porin trimer in the outer membrane was inferred from topological data on PhoE. According to this result a single channel of approx. 30 A diameter starts at the outer surface. Near the middle of the outer membrane bilayer this channel branches out into three separate channels, each running within a single porin monomer to the periplasmic surface.

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.

A protein with sequence identity to Skp (FirA) supports protein translocation into plasma membrane vesicles of Escherichia coli

We have purified to homogeneity a 15 kDa‐protein from a ribosomal salt extract of Escherichia coli that compensates in vitro a defect of SecA but not of SecB. Removal of this protein from a cell‐free transcription/translation system impairs translocation into plasma membrane vesicles of the precursors of LamB and to a lesser degree also of OmpA. These results suggest a role of the 15 kDa‐protein in bacterial protein export. The NH2‐terminal 35 amino acids were found to be identical to those of the skp (firA) gene product, to which several putative functions have previously been attributed.