The biological requirement of the trace element selenium was recognized 40 years ago. Selenium is incorporated into several enzymes and transfer RNA species of both prokaryotic and eukaryotic origin. In enzymes which contain a selenopolypeptide, selenium is present as covalently bound selenocysteine which participates in the catalytic reaction. Sequence analysis of the genes coding for two selenoproteins, formate dehydrogenase H from Escherichia coli and glutathione peroxidase from mouse and man, demonstrated that an in-frame UGA opal nonsense codon directs the incorporation of selenocysteine. In the case of formate dehydrogenase incorporation occurs cotranslationally. Recently, we identified four genes whose products are required for selenocysteine incorporation in E. coli. We report here that one of these genes codes for a tRNA species with unique properties. It possesses an anticodon complementary to UGA and deviates in several positions from sequences, until now, considered invariant in all tRNA species. This tRNA is aminoacylated with L-serine by the seryl-tRNA ligase which also charges cognate tRNASer. Selenocysteine, therefore, is synthesized from a serine residue bound to a natural suppressor tRNA which recognizes UGA.
HypF has been characterized as an auxiliary protein whose function is required for the synthesis of active [NiFe] hydrogenases in Escherichia coli and other bacteria. To approach the functional analysis, in particular the involvement in CO/CN ligand synthesis, HypF was purified from an overproducing strain to apparent homogeneity. The purified protein behaves as a monomer on size exclusion chromatography, and it is devoid of nickel or other cofactors. As indicated by the existence of a sequence motif also present in several O-carbamoyltransferases, HypF interacts with carbamoyl phosphate as a substrate and releases inorganic phosphate. In addition, HypF also possesses ATP cleavage activity that gives rise to AMP and pyrophosphate as products and that is dependent on the presence of carbamoyl phosphate. This and the fact that HypF catalyzes a carbamoyl phosphate-dependent pyrophosphate ATP exchange reaction suggest that the protein catalyzes activation of carbamoyl phosphate. Extensive mutagenesis of the putative functional motifs deduced from the derived amino acid sequence showed a full correlation of the resulting variants between their activity in hydrogenase maturation and the in vitro reactivity with carbamoyl phosphate. The results are discussed in terms of the involvement of HypF in the conversion of carbamoyl phosphate to the CN ligand.Hydrogen metabolism in enterobacteria involves the activity of the products of three functional classes of genes that code for structural proteins, regulatory proteins, or for proteins involved in metal center biosynthesis and enzyme maturation. In Escherichia coli, the structural genes are organized in four operons, responsible for the formation of hydrogenase 1 (hya operon), hydrogenase 2 (hyb operon), hydrogenase 3 (hyc operon), and the putative hydrogenase 4 (hyf operon). Each operon contains the genes for the large and small hydrogenase subunit, for redox carriers, membrane anchor proteins plus components required for the maturation of the hydrogenase encoded by that specific operon, like the endopeptidase involved in proteolytic processing of the large subunit (for review see Refs. 1 and 2). Because the hydrogenases in E. coli serve different physiological functions, the expression of these operons is differentially regulated. Hydrogenase 3, for example, which is a component of the formate hydrogen lyase system, is synthesized under fermentative conditions. Its formation requires the activity of the transcriptional activator FhlA and formate as inducer (3).Considerable efforts have been directed to understand the incorporation of the [NiFe] metal center into the large hydrogenase subunit. A scenario is emerging indicating that iron and nickel insertion proceeds separately, whereby the incorporation of iron together with its CO and CN ligands precedes that of nickel (1, 4, 5). The function of the HypA and HypB proteins has been related to nickel insertion because hypA and hypB mutations can be phenotypically complemented by inclusion of high nickel concentrations in t...
The selD gene from Escherichia coli, whose product is involved in selenium metabolism, has been cloned and sequenced. selD codes for a protein of 347 amino acids with a calculated molecular weight of 36,687. Analysis of the selD gene product through expression of the gene in the phage T7 promoter/polymerase system confirmed the predicted molecular weight of the protein. Gene disruption experiments demonstrated that the SelD protein is required both for the incorporation of selenium into the modified nucleoside 5-methylaminomethyl-2-selenouridine of tRNA and for the biosynthesis of selenocysteine from an L-serine residue esterbonded to tRNASe A. tRNASeA has been purified, aminoacylated with L-serine, and used as a substrate for the development of an in vitro system for selenocysteine biosynthesis. Efficient formation of selenocysteinyl-tRNAsA was achieved by using extracts in which both the selD and the selA gene products were overproduced. The results demonstrate that selenocysteine is synthesized from L-serine bound to tRNAUCA and they are in accord with SelD functioning as a donor of reduced selenium.
A detailed analysis of the expression of the sel genes, the products of which are necessary for the specific incorporation of selenium into macromolecules in Escherichia coli, showed that transcription was constitutive, being influenced neither by atrobiosis or anaerobiosis nor by the intracellular selenium concentration. The gene encoding the tRNA molecule which is specifically aminoacylated with selenocysteine (seiC) proved to be monocistronic. In contrast, the other three sel genes (seL4, -B, and -D) were shown to be constituents of two unlinked operons. The selA and selB geites formed one transcriptional unit (seL41)7, while selD was shown to be the central gene in an operon including two other genes, the promoter distal of which (topB) encodes topoisomerase HI. The promoter proximal gene (orfl83) was sequenced and shown to encode a protein consisting of 183 amino acids (Mr,20,059), the amino acid sequence of which revealed no similarity to any currently known protein. The products of the orfl83 and topB genes were required neither for selenoprotein biosynthesis nor for selenation of tRNAs. seL4B transcription was driven by a single, weak promoter; however, two major selD operon transcripts were identified. The longer initiated just upstream of the orfl83 gene, whereas the 5' end of the other mapped in a 116-bp nontranslated region between orfl83 and selD. Aerobic synthesis of all four sel gene products incited a reexamination of a weak 110-kDa selenopolypeptide which is produced under these conditions. The aerobic appearance of this 110-kDa selenopolypeptide was not a consequence of residual expression of the gene encoding the 110-kDa selenopolypeptide of the anaerobically inducible formate dehydrogenase N (FDHN) enzyme, as previously surmised, but rather resulted from the expression of a gene encoding a third, distinct selenopolypeptide in E. coli. A mutant strain no longer capable of synthesizing the 80-and 110-kDa selenopolypeptides of FDHH and FDHN, respectively, still synthesized this alternative 110-kDa selenopolypeptide which was present at equivalent levels in cells grown aerobically and anaerobically with nitrate. Furthermore, this strain exhibited a formate-and sel gene-dependent respiratory activity, indicating that it is probable that this selenopolypeptide constitutes a major component of the formate oxidase, an enzyme activity initially discovered in aerobically grown E. coli more than 30 years ago.The products of four genes have been identified as being essential for the incorporation of selenium into macromolecules in Escherichia coli (8,44). The selD gene encodes a protein which is required for incorporation of selenium into both protein and modified tRNAs (28), whereas the products of the other three genes are required for the synthesis of selenopolypeptides (29). The selC gene product is a tRNA (30) which is aminoacylated with serine, and this serine residue is converted to selenocysteine on the tRNA through the joint action of SelD (28) and selenocysteine synthase, the selA gene product ...
The expression of the pyruvate formate-lyase gene (pfl) is induced by anaerobic growth, and this is increased further by growth in pyruvate. Previous work has shown that anaerobic induction is strongly dependent on the activator FNR and partially dependent on a second transcription factor, ArcA, while pyruvate induction only required FNR. Anaerobic and pyruvate regulation both require the presence of a 5' nontranslated regulatory sequence which spans approximately 500 bp of DNA. A mobility shift assay was developed to identify proteins that bind to this regulatory region. Several binding activities were separated by heparin agarose chromatography, and one of these activities was characterized and shown to be integration host factor (IHF). Mobility shift and DNase I footprinting experiments defined a single IHF binding site in the pfl promoter-regulatory region. With pfl-lacZ fusions, it could be shown that introduction of a himD mutation abolished pyruvate-dependent induction of anaerobic expression in vivo. The same result was observed when the pfl IHF binding site was mutated. In addition, the partial anaerobic induction of expression found in an fnr strain was completely blocked in an fnr himD double mutant and in an fnr IHF binding site double mutant. Taken together, these data suggest that IHF is necessary for both pyruvate induction and the anaerobic induction mediated by ArcA.
Two-dimensional gel electrophoresis was used to examine the response of the cellular proteins of Escherichia coli to various anaerobic growth conditions and to the presence or absence of a functional Fnr protein. The steady-state levels of 125 polypeptides were found to vary in either a positive or negative manner, with many polypeptides being affected under a number of conditions. A large number (21) of the anaerobically inducible polypeptides were shown to be totally independent of the presence of Fnr while 22 were shown to be reduced in a fnr mutant under all anaerobic growth conditions tested. A total of 8 proteins were shown to be reduced in a fnr mutant only in aerobically grown cells indicating that the Fnr protein has a function in the presence of oxygen. This was further confirmed by the observation that 15 anaerobically inducible polypeptides were also found to show an increase in aerobically grown cells, however, only in a fnr strain. This latter finding implies that Fnr may also exhibit repressor function. This effect of Fnr-dependent repression was also observed with several polypeptides in anaerobically grown cells.
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 W WM, 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 W WM 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. ß
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