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A Böck

Publications and source records attributed to A Böck.

At least 19 recordsLinked to original sources
PubMed2005

[NiFe]-Hydrogenase maturation endopeptidase: structure and function.

Hydrogenase maturation endopeptidases catalyse the terminal step in the maturation of the large subunit of [NiFe]-hydrogenases. They remove a C-terminal extension from the precursor of the subunit, triggering a conformational switch that results in the bridging of the Fe and Ni atoms of the metal centre via the thiolate of a cysteine residue and in closure of the centre. This review summarizes what is known about the structure of the protein, its substrate specificity and its possible reaction mechanism.

Hydrogenase↗
PubMed2002

Metal insertion into NiFe-hydrogenases.

The synthesis and the insertion of the metallocentre of NiFe-hydrogenases is a complex process, in which seven maturation enzymes plus ATP, GTP and carbamoyl phosphate are involved. The review summarizes what is known about the properties and activities of these auxiliary proteins, and postulates a pathway along which maturation may take place.

Escherichia coli↗
PubMed2001

Selenium-dependent growth of Treponema denticola: evidence for a clostridial-type glycine reductase.

Assessment of the nutritional requirements of Treponema denticola disclosed a strict growth dependence on selenium. In vivo labeling of cells of this organism with (75)Se and electrophoretic analysis revealed three labeled bands, two of which were selenoproteins correlating in size with subunits A and B of glycine reductase. Antibodies directed against glycine- or betaine-reductase subunits of Eubacterium acidaminophilum specifically also reacted with proteins from cell lysates of T. denticola. Moreover, ORFs within the T. denticola genome sequence were found whose products display high sequence similarity to glycine-reductase subunits. These findings strongly support the notion that T. denticola ferments amino acids via the activity of glycine reductase, an enzyme previously thought to be restricted to gram-positive bacteria.

Amino Acid Oxidoreductases↗
PubMed2001

Fidelity of metal insertion into hydrogenases.

The fidelity of metal incorporation into the active center of hydrogenase 3 from Escherichia coli was studied by analyzing the inhibition of the maturation pathway by zinc and other transition metals. Hydrogenase maturation of wild-type cells was significantly affected only by concentrations of zinc or cadmium higher than 200 microM, whereas a mutant with a lesion in the nickel uptake system displayed a total blockade of the proteolytic processing of the precursor form into the mature form of the large subunit after growth in the presence of 10 microM Zn(2+). The precursor could not be processed in vitro by the maturation endopeptidase even in the presence of an excess of nickel ions. Evidence is presented that zinc does not interfere with the incorporation of iron into the metal center. Precursor of the large subunit accumulated in nickel proficient cells formed a transient substrate complex with the cognate endoprotease HycI whereas that of zinc-supplemented cells did not. The results show that zinc can intrude the nickel-dependent maturation pathway only when nickel uptake is blocked. Under this condition zinc appears to be incorporated at the nickel site of the large subunit and delivers a precursor not amenable to proteolytic processing since the interaction with the endoprotease is blocked.

Binding, Competitive↗
PubMed2001

The hydH/G Genes from Escherichia coli code for a zinc and lead responsive two-component regulatory system.

The hydH/G genes from Escherichia coli code for a two-component regulatory system that has been implicated in the regulation of hydrogenase 3 formation. In a detailed study of the function of HydH/G employing hycA'-'lacZ reporter gene fusions, it was shown that HydH/G indeed led to a stimulation of activation of the hycA promoter responsible for hydrogenase 3 synthesis but only when hydG is overexpressed from a plasmid in a strain lacking FhlA. Since the stimulation was not observed with an fdhF'-'lacZ fusion, and since it was independent from a functional hydH gene product, it must be considered as unspecific cross-talk. An extensive search for the actual physiological signal of HydH/G showed that the system responds to high concentrations of zinc or lead in the medium. Expression of zraP, a gene inversely oriented to hydH/G whose product seems to be involved in acquisition of tolerance to high Zn(2+) concentrations, is stimulated by high Zn(2+) and Pb(2+) concentrations and this stimulation requires both HydH and HydG. Purified HydG in the presence of phosphoryl donors binds to a region within the zraP-hydHG intergenic region that is characterised by two inverted repeats separated by a 14 bp spacer. Putative -12/-24 sigma(54)-dependent promoter motifs are present upstream of both the zraP and the hydHG transcriptional units; in accordance, transcription of zraP is strictly dependent on the presence of a functional rpoN gene. The expression of hydH/G is autoregulated: high Zn(2+) and Pb(2+) concentrations lead to a significant increase of the HydG protein content which took place only in a hydH(+) genetic background. Since HydH binds to membranes tightly, it is assumed that the HydH/G system senses high periplasmic Zn(2+) and Pb(2+) concentrations and contributes to metal tolerance by activating the expression of zraP. The redesignation of hydH/G as zraS/R is suggested.

Bacterial Proteins↗
PubMed2001

Carbamoylphosphate requirement for synthesis of the active center of [NiFe]-hydrogenases.

The iron of the binuclear active center of [NiFe]-hydrogenases carries two CN and one CO ligands which are thought to confer to the metal a low oxidation and/or spin state essential for activity. Based on the observation that one of the seven auxiliary proteins required for the synthesis and insertion of the [NiFe] cluster contains a sequence motif characteristic of O-carbamoyl-transferases it was discovered that carbamoyl phosphate is essential for formation of active [NiFe]-hydrogenases in vivo and is specifically required for metal center synthesis suggesting that it is the source of the CO and CN ligands. A chemical path for conversion of a carbamoyl group into cyano and carbonyl moieties is postulated

Amino Acid Motifs↗
PubMed2001

Functional analysis of prokaryotic SELB proteins.

Since the discovery of selenocysteine as the 21st amino acid considerable progress has been made in elucidating the system responsible for its insertion into proteins. Elongation factor SELB, whose amino-terminal part shows homology to EF-Tu, was found to be the key component mediating delivery of selenocysteyl-tRNA(Sec) to the ribosomal A site. It exhibits a distinct tertiary structure comprising binding sites for guanosine nucleotides, the cognate tRNA, an mRNA secondary structure (SECIS element) and presumably ribosomal components. The kinetics of interaction of SELB with its ligands have been studied in detail. GDP was found to bind with about 20-fold lower affinity than GTP and to be in rapid exchange, which obviates the need for a guanosine nucleotide exchange factor. The affinity of SELB for the SECIS element is in the range of 1 nM and further increases upon binding of selenocysteyl-tRNA(Sec) to the protein. This supports the model that SELB forms a tight quaternary complex on the SECIS element which is loosened after insertion of the tRNA into the ribosomal A site and the concomitant hydrolysis of GTP.

Bacterial Proteins↗
PubMed2001

Selenoprotein synthesis in archaea.

The availability of the genome sequences from several archaea has facilitated the identification of the encoded selenoproteins and also of most of the components of the machinery for selenocysteine biosynthesis and insertion. Until now, selenoproteins have been identified solely in species of the genera Methanococcus (M.) and Methanopyrus. Apart from selenophosphate synthetase, they include only enzymes with a function in energy metabolism. Like in bacteria and eukarya, selenocysteine insertion is directed by a UGA codon in the mRNA and involves the action of a specific tRNA and of selenophosphate as the selenium donor. Major differences to the bacterial system, however, are that no homolog for the bacterial selenocysteine synthase was found and, especially, that the SECIS element of the mRNA is positioned in the 3' nontranslated region. The characterisation of a homolog for the bacterial SelB protein showed that it does not bind to the SECIS element necessitating the activity of at least a second protein. The use of the genetic system of M. maripaludis allowed the heterologous expression of a selenoprotein gene from M. jannaschii and will facilitate the elucidation of the mechanism of the selenocysteine insertion process in the future.

Amino Acid Sequence↗
PubMed2001

Heterologous expression of archaeal selenoprotein genes directed by the SECIS element located in the 3' non-translated region.

Previous in silico analysis of selenoprotein genes in Archaea revealed that the selenocysteine insertion (SECIS) motif necessary to recode UGA with selenocysteine was not adjacent to the UGA codon as is found in Bacteria. Rather, paralogous stem-loop structures are located in the 3' untranslated region (3' UTR), reminiscent of the situation in Eukarya. To assess the function of such putative SECIS elements, the Methanococcus jannaschii MJ0029 (fruA, which encodes the A subunit of the coenzyme F420-reducing hydrogenase) mRNA was mapped in vivo and probed enzymatically in vitro. It was shown that the SECIS element is indeed transcribed as part of the respective mRNA and that its secondary structure corresponds to that predicted by RNA folding programs. Its ability to direct selenocysteine insertion in vivo was demonstrated by the heterologous expression of MJ0029 in Methanococcus maripaludis, resulting in the synthesis of an additional selenoprotein, as analysed by 75Se labelling. The selective advantage of moving the SECIS element in the untranslated region may confer the ability to insert more than one selenocysteine into a single polypeptide. Evidence for this assumption was provided by the finding that the M. maripaludis genome contains an open reading frame with two in frame TGA codons, followed by a stem-loop structure in the 3' UTR of the mRNA that corresponds to the archaeal SECIS element.

3' Untranslated Regions↗
PubMed2001

Interplay between the specific chaperone-like proteins HybG and HypC in maturation of hydrogenases 1, 2, and 3 from Escherichia coli.

The hybG gene product from Escherichia coli has been identified as a chaperone-like protein acting in the maturation of hydrogenases 1 and 2. It was shown that HybG forms a complex with the precursor of the large subunit of hydrogenase 2. As with HypC, which is the chaperone-like protein involved in hydrogenase 3 maturation, the N-terminal cysteine residue is crucial for complex formation. Introduction of a deletion into hybG abolished the generation of active hydrogenase 2 but only quantitatively reduced hydrogenase 1 activity since HypC could replace HybG in this function. In contrast, HybG could not take over the role of HypC in a DeltahypC genetic background. Overproduction of HybG, especially of the variants with the replaced N-terminal cysteine residue, strongly interfered with hydrogenase 3 maturation, apparently by titrating some other component(s) of the maturation machinery. The results indicate that the three hydrogenase isoenzymes not only are interacting at the functional level but are also interconnected during the maturation process.

Bacterial Proteins↗
PubMed2000

Identification and characterisation of the selenocysteine-specific translation factor SelB from the archaeon Methanococcus jannaschii.

Selenocysteine insertion into archaeal selenopolypeptides is directed through an mRNA structure (the SECIS element) situated in the 3' non-translated region like in eukaryotes. To elucidate the mechanism how this element affects decoding of an in-frame UGA with selenocysteine the open reading frames of the genome of Methanococcus jannaschii were searched for the existence of a homolog to the bacterial specialized translation factor SelB. The product of the open reading frame MJ0495 was identified as the archaeal SelB homolog on the basis of the following characteristics: (1) MJ0495 possesses sequence features characteristic of bacterial SelB; (2) purified MJ0495 displays guanine nucleotide binding properties like SelB; and (3) it preferentially binds selenocysteyl-tRNA(Sec). In contrast to bacterial SelB, however, no binding of MJ0495 protein to the SECIS element of the mRNA was found under the experimental conditions employed which correlates with the fact that MJ0495 lacks the C-terminal domain of the bacterial SelB protein known to bind the SECIS element. It is speculated that in Archaea the functions of bacterial SelB are distributed over at least two proteins, one, serving as the specific translation factor, like MJ0495, and another one, binding to the SECIS which interacts with the ribosome and primes it to decode UGA.

Amino Acid Sequence↗
PubMed2000

Dissection of the maturation reactions of the [NiFe] hydrogenase 3 from Escherichia coli taking place after nickel incorporation.

The steps in the maturation of the precursor of the large subunit (pre-HycE) of hydrogenase 3 from Escherichia coli taking place after incorporation of both iron and nickel were investigated. Pre-HycE could be matured and processed in the absence of the small subunit but association with the cytoplasmic membrane required heterodimer formation between the two subunits. Pre-HycE formed a complex with the chaperone-like protein HypC in the absence of the small subunit and, in this complex, also incorporated nickel. For the C-terminal processing, HypC had to leave the complex since only a HypC-free, nickel-containing form of pre-HycE was a substrate for the maturation endopeptidase.

Cell Fractionation↗
PubMed2000

Analysis of the cleavage site specificity of the endopeptidase involved in the maturation of the large subunit of hydrogenase 3 from Escherichia coli.

The maturation of [NiFe]-hydrogenases is a catalysed process in which the activities of at least seven proteins are involved. The last step consists of the endoproteolytic cleavage of the precursor of the large subunit after the [NiFe]-metal centre has been assembled. The amino acid sequence requirements for the endopeptidase HycI involved in the C-terminal processing of HycE, the large subunit of the hydrogenase 3 from Escherichia coli, were investigated. Mutational alteration of the amino acid residues neighbouring the cleavage site showed that proteolysis still occurred when chemically similar amino acids were exchanged. Processing was blocked, however, in a variant in which the methionine at the C-terminal side was replaced by a glutamate residue. Truncation of the precursor from the C-terminal end rendered variants amenable to maturation even when two-thirds of the extension were removed but abolished proteolysis upon further deletion of a cluster of six basic amino acids. A construct in which the C-terminal extension from the large subunit of the hydrogenase 2 was fused to the mature part of the large subunit of hydrogenase 3 was neither processed by HycI nor by HybD, the endopeptidase specific for the large subunit of hydrogenase 2. The maturation endopeptidase, therefore, exhibits a relaxed sequence constraint in recognition of its cleavage site and does not require the entire C-terminal extension. The results point to an interaction of the C-terminus with some domain of the large subunit, rendering a conformation amenable to recognition by the endopeptidase.

Amino Acid Sequence↗
PubMed2000

Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway.

A chromosomal fragment has been identified in a gene bank from Escherichia coli, which augmented the yield of cysteine in an industrial production strain. Subcloning and genetic analysis showed that an open reading frame coding for a product of 299 amino acids (Orf299) was responsible. Orf299 was synthesized in the T7 polymerase/promoter system and exhibited the properties of an integral membrane protein. Mutational interruption of orf299 did not cause a distinct phenotype; however, transformants overexpressing orf299 had lost the ability to grow in minimal medium unless it was supplemented with a source of reduced sulphur compounds, and they excreted considerable amounts of cysteine and O-acetyl-L-serine, especially in the presence of thiosulphate. Most of the cysteine was found to be masked in 2-methyl-2,4-thiazolidinedicarboxylic acid. N-acetyl-L-serine was also present in the medium, but it is open to question whether it represents a primary excretion product. Measurement of the induction status of the cysteine regulon by means of a cysK'-'lacZ gene fusion demonstrated that the regulon is not induced upon growth in the presence of a poor sulphur source and that the introduction of a constitutive cysB allele alleviates this deficiency. The results indicate that orf299 codes for an export pump for different metabolites of the cysteine pathway. Its relation to other efflux systems and the physiological role are discussed.

Amino Acid Sequence↗
PubMed2000

Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation.

The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino-acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydrogenases as a recognition motif.

Amino Acid Motifs↗
PubMed2000

Analysis of the domain structure and the DNA binding site of the transcriptional activator FhlA.

FhlA is the transcriptional activator of the genes coding for the formate hydrogen lyase system in Escherichia coli. It is activated by the binding of formate and induces transcription by sigma54 RNA polymerase after binding to specific upstream activating sequences (UAS). Sequence comparison had shown that FhlA exhibits a structure composed of three domains, which is typical for sigma54-dependent regulators. By analyzing the N-terminal domain of FhlA of E. coli (amino acids 1-378; FhlA-N) and the rest of the protein (amino acids 379-693; FhlA-C) as separate proteins in vivo and in vitro the functions of the different domains of FhlA were elucidated. The FhlA-C domain is active in ATP hydrolysis and activation of transcription and its activity is neither influenced by the presence of formate nor of the antiactivator HycA. However, it is stimulated in the presence of the FhlA-specific UAS, indicating that this region of FhlA is responsible for DNA binding. FhlA-N is not active itself but able to reduce the activity of full-length FhlA in trans, probably by formation of nonfunctional heterooligomers. The DNA binding site of FhlA was analyzed by hydroxyradical footprinting. Each UAS consists of two binding sites of 16 bp separated by a spacer region. A consensus sequence could be deduced and a model is presented and supported by in vivo data in which a FhlA tetramer binds to the UAS on one side of the DNA helix. Performing an extensive screening we could show that the FhlA regulatory system is conserved in different species of the family Enterobacteriaceae. The analysis of orthologs of FhlA revealed that they are able to functionally replace the E. coli enzyme.

Amino Acid Sequence↗