top of page

Benzylsuccinate synthase and other Fumarate adding enzymes (FAE)

All known organisms capable of anaerobic toluene metabolism initiate this pathway by the addition of the methyl group of toluene to a fumarate co-substrate to yield (R)-benzylsuccinate. This reaction represents a new biochemical principle and is catalysed by a glycyl-radical-enzyme, benzylsuccinate synthase (BSS). The main aim of this research is to elucidate the structure-function relation of FAE. We are investigating this question by focusing on mutating selected residues of the active site of benzylsuccinate synthase (BSS) in the large subunit and the Fe-S-cluster binding sites of the small subunits. The former residues are obviously involved in selecting and discriminating both substrates, toluene, and fumarate, as well as in enabling the catalytic mechanism, while the significance of the latter for the reaction is unknown. 

Reaction catalyzed by FAE enzymes - addition of fumarate to methyl carbon of various aromatic and aliphatic hydrocarbons.

​

​

An additional sub-project will deal with the recombinant expression and characterization of other natural variants of fumarate-adding enzymes, especially methylnaphthalene-, methylphenanthrene- and alkane-activating enzymes, including mutant variants. Many of these enzymes are not accessible in their native form, because their natural hosts grow too slowly and do not produce enough cell mass to allow their purification. Since we have obtained a recombinant expression system with apparent activation of BSS, we also intend to investigate the respective activating enzyme, aiming to establish in-vitro activation of FAE. In addition, we are aiming at the crystallization of the obtained new FAE to obtain a larger data basis for theoretical modeling and increasing their reaction ranges and application potentials.

We are conducting further quantum chemical modeling of the reaction mechanisms of FAE in parallel to experimental evaluations of the predictions to obtain more realistic models of their activities. Finally, we will aim at obtaining an efficient whole-cell recombinant system for biotechnological production of benzylsuccinate and its derivatives which may find future applications as chiral building blocks in the synthesis of fine chemicals or pharmaceutics or as components for polyester bioplastics.

​

This research is supported by NCN/DFG joint project Beethoven Life 2018/31/F/NZ1/01856 [2020-2023] "Structure and function of fumarate-adding glycyl radical enzymes: biochemistry, modeling and application (FAEREACTION)" 

​

List of the papers on the topic:

  1. M. Szaleniec, G. Oleksy, A. SekuÅ‚a, I. Aleksić, R. Pietras, M. Sarewicz, K. Krämer, A. J. Pierik, J Heider "Modeling the Initiation Phase of the Catalytic Cycle in the Glycyl-Radical Enzyme Benzylsuccinate Synthase", J. Phys. Chem B, 128, (2024) 5823-5839, DOI: 10.1021/acs.jpcb.4c01237

  2. I. Salii, M. Szaleniec, A. Alhaj Zein, D. Seyhan, A. SekuÅ‚a, K. Schühle, I. Kaplieva-Dudek, U. Linne, R. U. Meckenstock, J. Heider, "Determinants for Substrate Recognition in the Glycyl Radical Enzyme Benzylsuccinate Synthase Revealed by Targeted Mutagenesis", ACS Catalysis, 11 (2021) 3361-3370

  3. J. Heider, M. Szaleniec, B.M. Martins, D. Seyhan, W. Buckel, B.T. Golding, "Structure and Function of Benzylsuccinate Synthase and Related Fumarate-Adding Glycyl Radical Enzymes", J. Mol. Microbiol. Biotechnol., 26 (2016) 29-44

  4. M. Szaleniec, J. Heider, "Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate Synthase", Int. J. Mol. Sci. , 17 (2016) article number 514

  5. D. Seyhan, P. Friedrich, M. Szaleniec, M. Hilberg, W. Buckel, B. T. Golding, J. Heider, "Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene", Angew. Chem. Int. Ed., 5 (2016) 11664–11667

Fig. 1 Proposed reaction catalyzed by BSS and other FAE enzymes

bottom of page