Mechanistic and structural investigations into the unique monooxygenase CobG involved in vitamin B12 (cobalamin) biosynthesis

• Applicant: Dr. Rebekka Biedendieck
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2007 to 2010
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 58324451

• Final Report Year: 2010

Final Report Abstract

Vitamin B12 is a water soluble molecule which is an important nutrient for most eukaryotes. However, eukaryotes cannot synthesize B12 de novo. Vitamin B12 is essential for enzymes like the methylmalonyl CoA mutase and methionine synthase. Here, various enzymes of the two bacterial B12 biosynthesis pathways in the presence and absence of oxygen were biochemically characterized. The monooxygenase CobG involved in the O2-dependent pathway of vitamin B12 biosynthesis was found to have a [4Fe-4S] cluster as well as a non-heam iron to catalyze the reaction from precorrin-3A to precorrin-3B. This step provides the tetrapyrrole for the ring contraction. Although the [4Fe-4S] centre of CobG is O2- sensitive, the enzyme needs molecular oxygen for catalyzing its reaction. Another enzyme investigated in the frame work of this stipend was CobL. CobL is involved in the methylation of two C-atoms and one decarboxylation of precorrin 6B during the O2-dependent pathway. This enzyme was thought to be a fusion enzyme, consisting of CobL(E) and CobL(T), corresponding to the enzymes CbiE and CbiT of the O2-independent pathway. First results of in vivo assays seem to confirm the assumption. The lower ligand of vitamin B12 is 5,6-dimethylbenzimidazole (DMB). BluB catalyzes the reaction from FMN to form DMB. The BluB enzyme of B. megaterium, utilizing the O2-independent pathway to form vitamin B12, was detected and initially characterized. It is the first time that the enzyme providing the lower ligand of vitamin B12 in the O2- independent pathway was described. B. megaterium is know for its O2-independent biosynthesis of vitamin B12. As more than 30 enzymes are involved in the vitamin B12 pathway many different bottlenecks might occur. The B12 synthesis of B. megaterium was systematically optimized to increase the final vitamin B12 amount up to almost 40 fold.

Publications

• (2009) A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium. Appl Microbiol Biotechnol. 82: 1195-1203
  Gamer, M., Fröde, D., Biedendieck, R., Stammen, S. & Jahn D.
  
• (2009) Directed optimization of biocatalytic transglycosylation processes by the integration of genetic algorithms and fermentative approaches into a kinetic model. Process Biochemistry 44: 1103-1114
  Beine, R., Valente, A.R., Biedendieck, R., Jahn, D., & Seibel, J.
  
• Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center. J Biol Chem 284: 4796-4805
  Schroeder, S., Lawrence, A.D., Biedendieck, R., Rose, R.S., Deery, E., Graham, R.M., McLean, K.J., Munro, A.W., Rigby, S.E. & Warren, M.J.
  
• (2010) A short story about a big magic bug. Bioengineered Bugs 1: 1-7
  Bunk, B., Schulz, A., Stammen, S., Münch, R., Warren, M. J., Rohde, M., Jahn, D. & Biedendieck, R.
  
• (2010) Heme biosynthesis is coupled to electron transport chains for energy generation. Proc. Natl. Acad. Sci. USA 107: 10436-10441
  Moebius, K., Arias-Cartin, R., Breckau, D., Haennig, A.-L., Riedmann, K., Biedendieck, R., Schroeder, S., Becher, D., Magalon, A., Moser, J., Jahn, M. & Jahn, D.
  
• (2010) High yield intra- and extracellular protein production using Bacillus megaterium. Appl. Environ. Microbiol. 76: 4037-4046
  Stammen, S., Müller, B.K., Korneli, C., Biedendieck, R., Gamer, M., Franco- Lara, E. & Jahn, D.
  
• (2010) Industrial production by Bacillus megaterium and other Bacilli. Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology 1-15
  Bunk, B., Biedendieck, R., Jahn, D. & Vary, P.S.
  
• (2010) Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium. Microbial Biotechnology 3: 24-37
  Biedendieck, R., Malten, M., Barg, H., Bunk, B., Martens, J.-H., Deery, E., Leech, H., Warren, M. J. & Jahn, D.
  
• (2010) Systems biology of recombinant protein production in Bacillus megaterium in "Advances in Biochemical Engineering / Biotechnology” (Scheper, T., Ed.); Vol. 113 - Biosystems Engineering (Wittmann, C., Ed.) Springer, Berlin, Heidelberg 120: 133-161
  Biedendieck, R., Bunk, B., Fürch, T., Franco-Lara, E., Jahn, M. & Jahn, D.
  
• Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium (2010). Appl. Microbiol. Biotechnol. 88: 529-539
  Stammen, S., Schuller, F., Dietrich, S., Gamer, M., Biedendieck, R. & Jahn, D.