More Theory about our Approach
The blueprint of our B12 Synporter
Our B
12 Synporter is built of a B
12-binding protein domain linked to a signal for a Twin Arginine Transporter (Tat) mediated export. While the B
12-binding domain (obviously) binds the vitamin, the export signal allows the protein-vitamin complex to leave the cytoplasm via the Tat system. In the following section, we will have a further look on these components.
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). Therefore, our B
12-bound Synporter should be able to be exported using this pathway.
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 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. 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). For our Synporter we chose the TorA signal peptide from 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). To connect the export signal with a B
12-binding protein domain we use the five amino acids following the signal peptide in trimethylamine-N-oxide reductase as a linker.
The B
12 binding domain
For the binding of B
12 to our Synporter, we use domains of proteins which are naturally capable of doing so. To increase our chances of success, we chose to test three different constructs.
MutB is the α-subunit of the methylmalonyl coenzyme A mutase from the Gram-positive bacterium Propionibacterium shermanii. The enzyme family it belongs to uses adenosylcobalamin to create a substrate radical that then rearranges to exchange a hydrogen atom with a group attached to a neighbouring carbon atom. Methylmalonyl CoA mutase catalyses the interconversion between linear succinyl CoA and branched methylmalonyl CoA and is thus involved in the fermentation of pyruvate to propionate (Allen et al.,1963). It is an αβ heterodimer and has a molecular weight of 150 kDa (80 kDa for the α chain and 70 kDa for the β chain). The active site as well as the cobalamin binding site are located in the α chain (Francalanci et al., 1986). Crystal structures of the whole enzyme bound to cobalamin (PDB 1REQ; Mancia et al., 1996) as well as bound to cobalamin and its substrate (PDB 4REQ; Manica and Evans, 1998) are available. Since a TorA-mutB fusion protein could already be produced in significant amounts and has been exported to the periplasm in E. coli BL21, C. freundii and E. blattae (Toeche-Mittler, 2002), MutB is our most promising candidate.
GlmS is the B
12-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 B
12. While GlmS as the smaller subunit (14.8 kDa) binds B
12, 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 (PDB 1I9C; Gruber et al., 2001) as well as an NMR structure of only GlmS (PDB 1B1A; Hoffmann et al., 1999) are available.
BtuF is the periplasmic binding protein for the vitamin B
12 transporter BtuCD from E. coli (Cadieux et al., 2002). While some bacteria and archaea are capable of its synthesis, E. coli belongs to the majority of prokaryotes that contain transport systems to import B
12 (Warren et al., 2002). Its transmembrane transport is achieved by the Btu (B twelve uptake) system composed of BtuB, an outer membrane TonB-dependent transporter (Cadieux et al., 1999), and the ABC transporter BtuCDF, which is located in the inner membrane. While BtuC and BtuD compose respectively the trans-membrane domain and the ABC (Bassford et al., 1977), BtuF is the periplasmic binding protein. It has a size of 30.19 kDa and is composed of two globular domains, between which vitamin B
12 is bound, linked by a rigid interdomain α-helix (Karpowich et al., 2003). A crystal structure of the protein bound to B
12 is available (PDB1N4A; Karpowich et al., 2003). For us it is highly interesting to see, if a protein that is naturally involved in B
12 import can be converted to mediate its export. If this was possible, the concept could potentially be applied to other proteins involved in the import of small molecules.
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