Markus J. Bröcker

Recoding the genetic code with selenocysteine.

Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as the Sec insertion machinery was found to be able to site-specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid (AA) to Sec was observed along with a tenfold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high-fidelity sense-codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl-tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell.

Decameric SelA•tRNASec Ring Structure Reveals Mechanism of Bacterial Selenocysteine Formation

Putting Selenium in Proteins The 21st amino acid, selenocysteine (Sec), occurs in the active site of many redox enzymes. Its cognate transfer RNA (tRNA) is first loaded with Ser by seryl-tRNA synthetase and the Ser-tRNASec is then converted to Sec-tRNASec. Itoh et al. (p. 75) determined the crystal structures of the selenocysteine synthase, SelA, that is responsible for this conversion in bacteria, alone and in complex with tRNA. The decameric SelA complex binds to 10 tRNASec molecules. The structures, together with biochemistry, show how SelA discriminates tRNASec from tRNASer, give insight into the mechanism of catalysis, and show that decamerization is essential for function. Structural and biochemical data reveal how selenocysteine is produced from serine on transfer RNA. The 21st amino acid, selenocysteine (Sec), is synthesized on its cognate transfer RNA (tRNASec). In bacteria, SelA synthesizes Sec from Ser-tRNASec, whereas in archaea and eukaryotes SepSecS forms Sec from phosphoserine (Sep) acylated to tRNASec. We determined the crystal structures of Aquifex aeolicus SelA complexes, which revealed a ring-shaped homodecamer that binds 10 tRNASec molecules, each interacting with four SelA subunits. The SelA N-terminal domain binds the tRNASec-specific D-arm structure, thereby discriminating Ser-tRNASec from Ser-tRNASer. A large cleft is created between two subunits and accommodates the 3′-terminal region of Ser-tRNASec. The SelA structures together with in vivo and in vitro enzyme assays show decamerization to be essential for SelA function. SelA catalyzes pyridoxal 5′-phosphate–dependent Sec formation involving Arg residues nonhomologous to those in SepSecS. Different protein architecture and substrate coordination of the bacterial enzyme provide structural evidence for independent evolution of the two Sec synthesis systems present in nature.

Rewiring translation for elongation factor Tu-dependent selenocysteine incorporation

Selenium is an essential micronutrient for animals.[1] Humans contain 25 presumably essential selenoproteins[2] in which selenium is found in the form of Sec.[3] In this 21st genetically encoded amino acid[4] the thiol moiety of Cys is replaced by a selenol group. In all Sec-decoding organisms, Sec biosynthesis (Scheme 1B) starts with the acylation of tRNASec by seryl-tRNA synthetase (SerRS) to form Ser-tRNASec (reviewed in[5]). In bacteria, conversion of Ser-tRNASec to Sec-tRNASec is achieved by SelA (reviewed in[4]). In contrast, archaea and eukaryotes employ an additional phosphorylation step. O-phosphoseryl-tRNASec kinase (PSTK) phosphorylates the tRNA-bound Ser moiety of Ser-tRNASec to form O-phosphoseryl-tRNASec (Sep-tRNASec),[6] the substrate for SepSecS that catalyzes the tRNA-dependent Sep to Sec conversion.[7] The selenium donor for both SelA and SepSecS is selenophosphate (reviewed in[4, 7b]).

A synthetic tRNA for EF-Tu mediated selenocysteine incorporation in vivo and in vitro

Incorporation of selenocysteine (Sec) in bacteria requires a UGA codon that is reassigned to Sec by the Sec‐specific elongation factor SelB and a conserved mRNA motif (SECIS element). These requirements severely restrict the engineering of selenoproteins. Earlier, a synthetic tRNASec was reported that allowed canonical Sec incorporation by EF‐Tu; however, serine misincorporation limited its scope. We report a superior tRNASec variant (tRNAUTuX) that facilitates EF‐Tu dependent stoichiometric Sec insertion in response to UAG both in vivo in Escherichia coli and in vitro in a cellfree protein synthesis system. We also demonstrate recoding of several sense codons in a SelB supplemented cell‐free system. These advances in Sec incorporation will aid rational design and directed evolution of selenoproteins.

Iron-sulfur cluster-dependent catalysis of chlorophyllide a oxidoreductase from Roseobacter denitrificans.

Background: Synthesis of bacteriochlorophylls is essential for the photosynthetic capture of solar energy. Results: Chlorophyllide a oxidoreductase (COR) performs nitrogenase-related redox catalysis using two cysteine-ligated [4Fe-4S] clusters. Conclusion: A reductive trans protonation mechanism and an identical substrate binding mode for nitrogenase-like enzymes was deduced. Significance: Energy transduction of COR is relevant for related systems involved in the reduction of chemically stable multibonds. Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)2 with the catalytic (BchY/BchZ)2 protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF4−. Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)2. A second [4Fe-4S] cluster was identified on (BchY/BchZ)2. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.

Dimer-Dimer Interaction of the Bacterial Selenocysteine Synthase SelA Promotes Functional Active-Site Formation and Catalytic Specificity

The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA(Sec)). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA(Sec), formed by seryl-tRNA synthetase, to Sec-tRNA(Sec). SelA, a member of the fold-type-I pyridoxal 5-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA(Sec) revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA(Sec). The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of depentamerized SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.