Karl Forchhammer

Selenocysteine: the 21st amino acid

Great excitement was elicited in the field of selenium biochemistry in 1986 by the parallel discoveries that the genes encoding the selenoproteins glutathione peroxidase and bacterial formate dehydrogenase each contain an in‐frame TGA codon within their coding sequence. We now know that this codon directs the incorporation of selenium, in the form of selenocysteine, into these proteins. Working with the bacterial system has led to a rapid increase in our knowledge of selenocysteine biosynthesis and to the exciting discovery that this system can now be regarded as an expansion of the genetic code. The prerequisites for such a definition are co‐translational insertion into the polypeptide chain and the occurrence of a tRNA molecule which carries selenocysteine. Both of these criteria are fulfilled and, moreover, tRNASec even has its own special translation factor which delivers it to the translating ribosome. It is the aim of this article to review the events leading to the elucidation of selenocysteine as being the 21st amino acid.

Selenoprotein synthesis: an expansion of the genetic code

A number of enzymes employ the unusual amino acid selenocysteine as part of their active site because of its high chemical reactivity. Selenocysteine is incorporated into these proteins co-translationally: biosynthesis occurs on a specific tRNA and insertion into a growing polypeptide is directed by a UGA codon in the mRNA. In E. coli, this requires a specific translation factor. Selenocysteine thus represents a unique expansion of the genetic code.

Identification of a novel translation factor necessary for the incorporation of selenocysteine into protein

DURING the biosynthesis of selenoproteins in both prokaryotes and eukaryotes, selenocysteine is cotranslationally incorporated into the nascent polypeptide chain1, 2 through a process directed by a UGA codon that normally functions as a stop codon3–5. Recently, four genes have been identified whose products are required for selenocysteine incorporation in Escherichia coli6. One of these genes,selC, codes for a novel transfer RNA species (tRNAUCA) that accepts serine and cotranslationally inserts selenocysteine by recognizing the specific UGA codon7. The serine residue attached to this tRNA is converted to selenocysteine in a reaction dependent on functional selA and selD gene products8. By contrast, the selB gene product (SELB) is not required until after selenocysteyl-tRNA biosynthesis8. Here we present evidence indicating that SELB is a novel translation factor. The deduced amino-acid sequence of SELB exhibits extensive homology with the sequences of the translation initiation factor-2 (IF-2) and elongation factor Tu (EF-Tu). Furthermore, purified SELB protein binds guanine nucleotides in a 1:1 molar ratio and specifically complexes selenocysteyl-tRNAUCA, but does not interact with seryl-tRNAUCA. Thus, SELB could be an amino acid-specific elongation factor, replacing EF-Tu in a special translational step.

Nitrogen-starvation-induced chlorosis in Synechococcus PCC 7942 : adaptation to long-term survival

When deprived of essential nutrients, the non-diazotrophic cyanobacterium Synechococcus sp. strain PCC 7942 undergoes a proteolytic degradation of the phycobiliproteins, its major light-harvesting pigments. This process is known as chlorosis. This paper presents evidence that the degradation of phycobiliproteins is part of an acclimation process in which growing cells differentiate into non-pigmented cells able to endure long periods of starvation. The time course of degradation processes differs for various photosynthetic pigments, for photosystem I and photosystem II activities and is strongly influenced by the illumination and by the experimental conditions of nutrient deprivation. Under standard experimental conditions of combined nitrogen deprivation, three phases of the differentiation process can be defined. The first phase corresponds to the well-known phycobiliprotein degradation, in phase 2 the cells lose chlorophyll a prior to entering phase 3, the fully differentiated state, in which the cells are still able to regenerate pigmentation after the addition of nitrate to the culture. An analysis of the protein synthesis patterns by two-dimensional gel electrophoresis during nitrogen starvation indicates extensive differential gene expression, suggesting the operation of tight regulatory mechanisms.

DEPHOSPHORYLATION OF THE PHOSPHOPROTEIN PII IN SYNECHOCOCCUS PCC 7942 : IDENTIFICATION OF AN ATP AND 2-OXOGLUTARATE-REGULATED PHOSPHATASE ACTIVITY

The phosphorylation state of the putative signal transduction protein PII from the cyanobacterium Synechococcus sp. strain PCC 7942 depends on the cellular state of nitrogen and carbon assimilation. In this study, dephosphorylation of phosphorylated PII protein (PII‐P) was investigated both in vivo and in vitro. The in vivo studies implied that PII‐P dephosphorylation is regulated by inhibitory metabolites involved in the glutamine synthetase–glutamate synthase pathway of ammonium assimilation. An in vitro assay for PII‐P dephosphorylation was established that revealed a Mg2+‐dependent PII‐P phosphatase activity. PII‐P phosphatase and PII kinase activities could be separated biochemically. A partially purified PII‐P phosphatase preparation also catalysed the dephosphorylation of phosphoserine/phosphothreonine residues on other proteins in a Mg2+‐dependent manner. However, only dephosphorylation of PII‐P was regulated by synergistic inhibition by ATP and 2‐oxoglutarate. As the same metabolites stimulate the PII kinase activity, it appears that the phosphorylation state of PII is determined by ATP and 2‐oxoglutarate‐dependent reciprocal reactivity of PII towards its phosphatase and kinase.

Heterotrimerization of PII‐like signalling proteins: implications for PII‐mediated signal transduction systems

PII‐like signalling molecules are trimeric proteins composed of 12–13u2003kDa polypeptides encoded by the glnB gene family. Heterologous expression of a cyanobacterial glnB gene in Escherichia coli leads to an inactivation of E. colis own PII signalling system. In the present work, we show that this effect is caused by the formation of functionally inactive heterotrimers between the cyanobacterial glnB gene product and the E. coli PII paralogues GlnB and GlnK. This led to the discovery that GlnK and GlnB of E. coli also form heterotrimers with each other. The influence of the oligomerization partner on the function of the single subunit was studied using heterotrimerization with the Synechococcus PII protein. Uridylylation of GlnB and GlnK was less efficient but still possible within these heterotrimers. In contrast, the ability of GlnB‐UMP to stimulate the adenylyl‐removing activity of GlnE (glutamine synthetase adenylyltransferase/removase) was almost completely abolished, confirming that rapid deadenylylation of glutamine synthetase upon nitrogen stepdown requires functional homotrimeric GlnB protein. Remarkably, however, rapid adenylylation of glutamine synthetase upon exposing nitrogen‐starved cells to ammonium was shown to occur in the absence of a functional GlnB/GlnK signalling system as efficiently as in its presence.

The Synechococcus Strain PCC 7942 glnN Product (Glutamine Synthetase III) Helps Recovery from Prolonged Nitrogen Chlorosis

We report the cloning and sequencing of the glnN gene encoding a class III glutamine synthetase from the cyanobacterium Synechococcus strain PCC 7942. Mapping of the transcriptional start site revealed a DNA sequence in the promoter region that resembles an imperfect NtcA binding motif. Expression of glnN is impaired in NtcA- and P(II)-deficient mutants. The only parameter which was negatively affected in the glnN mutant compared to the wild type was the recovery rate of prolonged nitrogen-starved cells with low concentrations of combined nitrogen.

The PII Protein in Synechococcus PCC 7942 Senses and Signals 2-Oxoglutarate Under ATP-Replete Conditions

The glnBgene product, termed PII protein, is widely distributed among bacteria and functions as a signal transduction protein in the central regulation of nitrogen metabolism (reviewed in 9). In proteobacteria, PII is modified by uridylylation at a conserved tyrosyl residue (Tyr 51); under nitrogen replete conditions, PII is present in its unmodified state whereas PII-UMP signals nitrogen-deficiency. Recently the 3-D structure of PII from Escherichia colihas been resolved (1), showing that the site of modification is located at the apex of a large solvent-protruding loop, termed T-loop. In contrast to proteobacteria, PII in cyanobacteria is not modified by uridylylation but is phosphorylated at a seryl residue (Ser 49) separated only by one amino acid from the conserved tyrosyl residue (2,4). This indicates that phosphorylation also occurrs at the solvent-exposed T-loop. In SynechococcusPCC 7942, the trimeric PII protein was shown to be involved in the coordination of carbon and nitrogen assimilation, in particular mediating the dependence on CO2 fixation for nitrate utilization (3). In vivo analyses revealed that the phosphorylation state of PII responds to the status of nitrogen and carbon assimilation (2,3). In the presence of ammonium, PIIis predominantly present in its unmodified form. In nitrate grown cells, the extent of PII phosphorylation depends on the CO2 supply to the cells: Under CO2-limiting conditions, only a low degree of PII phosphorylation is observed whereas under CO2 sufficiency, PII is efficiently phosphorylated. The highest level of phosphorylation is found in nitrogen-starved cells. Studying the PII modification system, therefore, offers the possibility to investigate a mechanism used by cyanobacteria to sense the environmental changes in the nitrogen and carbon supply.

Biologie und Biochemie des Elements Selen

The importance of selenium as an essential trace element has progressively emerged during the last years due to the analysis of selenium deficiency diseases and to the identification and characterization of a number of selenoenzymes. Selenium is incorporated in the catalytic site of the enzymes as an integral selenocysteine residue. The pathway of selenocysteine biosynthesis and incorporation has been elucidated recently for Escherichia coli. This article presents an overview on these subjects and describes the mechanisms which confer selenocysteine specificity in the framework of protein biosynthesis. In addition, some considerations concerning the phylogeny of selenocysteine incorporation are presented and a model for the evolution of the selenocysteine pathway is proposed.

Interspecies compatibility of selenoprotein biosynthesis in Enterobacteriaceae

Several species of Enterobacteriaceae were investigated for their ability to synthesise selenium-containing macromolecules. Selenated tRNA species as well as selenated polypeptides were formed by all organisms tested. Two selenopolypeptides could be identified in most of the organisms which correspond to the 80 kDa and 110 kDa subunits of the anaerobicaly induced formate dehydrogenase isoenzymes of E coli. In those organisms possessing both isoenzymes, their synthesis was induced in a mutually exclusive manner dependent upon whether nitrate was present during anaerobic growth. The similarity of the 80 kDa selenopolypeptide among the different species was assessed by immunollogical and genetic analyses. Antibodies raised against the 80 kDa selenopolypeptide from E. coli cross-reacted with an 80 kDa polypeptide in those organisms which exhibited fermentative formate dehydrogenase activity. These organisms also contained genes which hydridised with the fdhF gene from E. coli. In an attempt to identify the signals responsible for incorporation of selenium into the selenopolypeptides in these organisms we cloned a portion of the fdhF gene homologue from Enterobacter aerogenes. The nucleotide sequence of the cloned 723 bp fragment was determined and it was shown to contain an in-frame TGA (stop) codon at the position corresponding to that present in the E. coli gene. This fragment was able to direct incorporation of selenocysteine when expressed in the heterologous host, E. coli. Moreover, the E. coli fdhF gene was expressed in Salmonella typhimurium, Serratia marcescens and Proteus mirabilis, indicating a high degree of convervation of the selenating system throughout the enterobacteria.