Difference between revisions of "Part:BBa K1978001"

Revision as of 16:07, 20 October 2016

TorA-GlmS

The TorA-GlmS Biobrick consists of a TorA signal sequence linked to GlmS, protein capable of binding vitamin B₁₂. The TorA signal peptide allows export of fully-folded proteins through the inner membrane via the Tat (Twin-Arginine translocation) system. This construct thus enables export of vitamin B₁₂ bound to BtuF out of the cytoplasm. The TorA sequence codes for an amino-terminal signal peptide that harbours a twin-arginine motif which is vital for the recognition by the Tat system. The TorA signal sequence and the sequence coding for BtuF are connected by a linker of 15 bases, coding for the five amino acids following the signal peptide in trimethylamine-N-oxide reductase from E.coli.

Usage and Biology

GlmS

GlmS is the B₁₂-binding subunit of glutamate mutase (Glm) from Clostridium cochlearium. The mechanism by which the enzyme uses adenosylcobalamin is highly similar to methylmalonyl coenzyme A mutase. Glm catalyzes the reversible rearrangement of (2S)-glutamate to (2S,3S)-3-methylaspartate (Leutbecher et al., 1992). This reaction is the first step in the fermentation of glutamate to acetate and butyrate (Buckel et al., 1974). The assembly of the active enzyme, an α2β2 tetramer, is mediated by coenzyme B₁₂. While GlmS as the smaller subunit (14.8 kDa) binds B₁₂, the larger subunit GlmE harbors the substrate binding site (Zelder et al., 1994). Crystal structures of the whole enzyme in complex with its substrate and adenosylcobalamin ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1I9C PDB1I9C]; Gruber et al., 2001) as well as an NMR structure of only GlmS ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1B1A PDB1B1A]; Hoffmann et al., 1999) are available.

The Tat export pathway and its signal peptide

In bacteria and archaea, proteins located outside the cytoplasm can reach their destination via either the Sec or the Tat (twin-arginine translocation) export pathway. While the Sec system translocates proteins in an unstructured state, the Tat apparatus has the unusual property of transporting fully folded proteins (Palmer and Berks, 2012). This system is very flexible in regard to the types of proteins that can be exported and the number of exported proteins highly differs between organisms. The E.coli Tat system is capable of transporting substrates up to 70 Å in diameter (Berks et al., 2000). Many exported proteins containing non-covalently bound cofactors use this pathway, because the cofactor is held in place by the protein folding. The Tat pathway is only used by proteins containing certain types of cofactors that are classified as metal-sulphur clusters or nucleotide based cofactors, which include among others also cobalamins (Berks et al., 2003).

Proteins are targeted to the Tat apparatus by amino-terminal signal peptides that are normally cleaved by an externally facing signal peptidase (Lüke et al., 2009). The cleavage is mediated by an AxA motif. The overall architecture is similar to Sec signal peptides and includes a tripartite structure with a basic n region at the N terminus, a hydrophobic h region in the middle and a polar c region at the C terminus. The key element of a Tat signal peptide is the highly conserved twin-arginine motif, defined as SRRXFLK (see figure 1). The two arginines are almost always invariant, while the other residues occur with a frequency of > 50 %. The amino acid at position X is usually polar (Palmer and Berks, 2012).

The TorA signal peptide used in this biobrick belongs to the trimethylamine-N-oxide reductase from E.coli, a well characterized protein exported via the Tat system. Using this signal peptide, it has already been achieved to export a heterologous protein normally transported by the Sec pathway through the Tat system (Christóbal et al., 1999).

Figure 1. Schematic view of the Tat signal peptide aligned with signal sequences from proteins exported via the tat pathway.

Residues that match the Tat consensus are shown in red, with the twin arginines underlined.

Sequence and Features