Relevance

An adult needs approximately 3.0 µg Vitamin B₁₂ per day which is essential for certain functions: it is involved in processes concerning synthesis of DNA, hormones and neurotransmitters and is involved in the formation of the nervous system and blood. Hence, a B₁₂ deficiency can cause diverse diseases like cancer, dementia, depression, pernicious anemia and polyneuropathy.

Nowadays, many people do not eat meat or animals products at all. Therefore, these people have a high risk to suffer from B₁₂ deficiency if their diet does not provide enough B₁₂.

Vitamin B₁₂ is also used in the industry as it is needed in the biotechnical production of various organic substances. Furthermore, it is added to diverse daily products like toothpaste, fruit gum, non-diary milk or cleaning solution for contact lenses.

Our Aim

We intend to design, construct and introduce a synthetic Vitamin B₁₂ exporter (“Synporter”) into a production organism. Thereby, we aim for facilitated and higher yields in the industrial Vitamin B₁₂ production without requiring cell lysis. The B₁₂ Synporter consists of a signal for a Twin Arginine Transporter (Tat) mediated export that is linked to a B₁₂-binding domain. This construct will be expressed in S. typhimurium TA100, R. planticola and S. blattae. We will not only test these different production organisms but also different B₁₂-binding domains.

The B₁₂ binding domain

For the binding of B₁₂ 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 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 (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 B₁₂ 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 B₁₂ (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 B₁₂ is bound, linked by a rigid interdomain α-helix (Karpowich et al.., 2003). A crystal structure of the protein bound to B₁₂ is available (PDB1N4A; Karpowich et al.., 2003). For us it is highly interesting to see, if a protein that is naturally involved in B₁₂ 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.

References

1. Allen S. H. P., Kellermeyer R. W., Stjernholm R., Wood H. G. 1964 Purification and properties of enzymes involved in the propionic acid fermentation. J. Bacteriol. 87:171-187
2. Bassford P. J., Jr., Kadner R. J. 1977 Genetic Analysis of Components Involved in Vitamin B₁₂ Uptake in Escherichia coli. J. Bacteriol. 132:796–805.
3. Berks B. C., Sargent F., Palmer T. 2000 The Tat protein export pathway. Mol Microbiol. 35(2):260-74.
4. Berks, B. C., Palmer, T., Sargent, F. 2003 The Tat protein translocation pathway and its role in microbial physiology. Adv. Microb. Physiol. 47:187–254.
5. Buckel, W. & Barker, H.A. 1974 Two pathways of glutamate fermentation by anaerobic bacteria. J. Bacteriol. 117, 1248±1260.
6. Cadieux N, Bradbeer C, Reeger-Schneider E, Köster W, Mohanty AK, Wiener MC, Kadner RJ. 2002 Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli. J Bacteriol. 184(3):706-17.
7. Cadieux N., Kadner R. J. 1999 Site-directed disulfide bonding reveals an interaction site between energy-coupling protein TonB and BtuB, the outer membrane cobalamin transporter. Proc. Natl. Acad. Sci. U. S. A. 96:10673–10678.
8. Christόbal S., de Gier J.-W., Nielsen H. and von Heijne G., 1999 Competition between Sec- and Tat- dependent protein translocation in Escherichia coli. EMBO J. Vol. 18, No. 11: 2982-2990.
9. Francalanci F., Davis N. K., Fuller J. Q., Murfitt D., Leadlay P. F., 1986 The subunit structure of methylmalonyl-CoA mutase from Propionibacterium shermanii. Biochem. J. 236:489-494
10. Gruber, K., Reitzer, R., Kratky, C. 2001 Radical Shuttling in a Protein: Ribose Pseudorotation Controls Alkyl-Radical Transfer in the Coenzyme B(12) Dependent Enzyme Glutamate Mutase. Angew.Chem.Int.Ed.Engl. 40: 3377-3380
11. Hoffmann B, Konrat R, Bothe H, Buckel W, Kräutler B. 1999 Structure and dynamics of the B₁₂-binding subunit of glutamate mutase from Clostridium cochlearium. Eur. J. Biochem. 263(1):178-88.
12. Karpowich N. K., Huang H. H., Smith P. C., Hunt J. F. 2003 Crystal Structures of the BtuF Periplasmic-binding Protein for Vitamin B₁₂ Suggest a Functionally Important Reduction in Protein Mobility upon Ligand Binding. The Journal of Biological Chemistry 278, 8429-8434.
13. Leutbecher, U., Boecher, R., Linder, D. & Buckel, W. 1992 Glutamate mutase from Clostridium cochlearium. Purification, cobamide content and stereospecific inhibitors. Eur. J. Biochem. 205, 759±765.
14. Lüke, I., Handford, J. I., Palmer, T. & Sargent, F. 2009 Proteolytic processing of Escherichia coli twin-arginine signal peptides by LepB. Arch. Microbiol. 191:919–925.
15. Manica F., Evans P. R., 1998 Conformational changes on substrate binding to methylmalonyl CoA mutase and new insights into the free radical mechanism. Structure 6:711-720
16. Mancia, F., Keep, N.H., Nakagawa, A., Leadlay, P.F., McSweeney, S., Rasmussen, B., Bosecke, P., Diat, O., Evans, P.R. 1996 How coenzyme B₁₂ radicals are generated: the crystal structure of methylmalonyl-coenzyme A mutase at 2 A resolution. Structure 4:339-350
17. Palmer T., Berks B. C., 2012 The twin-arginine translocation (Tat) protein export pathway. Mature Reviews Microbiology 10:483-496
18. Toeche-Mittler, C. 2002 Konstruktion eines bakteriellen Systems zum Export von Coenzym B₁₂. Dissertation zum Erlangen des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen
19. Zelder, O., Beatrix, B., Leutbecher, U. & Buckel, W. 1994 Characterization of the coenzyme-B₁₂-dependent glutamate mutase from Clostridium cochlearium produced in Escherichia coli. Eur. J. Biochem. 226, 577±585.
20. Warren M. J., Raux E., Schubert H. L., Escalante-Semerena J. C. 2002 The biosynthesis of adenosylcobalamin (vitamin B₁₂). Nat. Prod. Rep. 19:390–412.