The rational based mutation opening new substrates to the glycil-radical enzyme.
Updated: Mar 4
nzymes accomplish many reactions that don't work with the methods of organic chemistry. An interesting example of this is the formation of C-C bonds between non-activated hydrocarbons and unsaturated compounds, e.g. fumarate. These reactions are catalyzed by a subgroup of glycyl radical enzymes, the fumarate-adding enzymes, which play a key role in the anaerobic bacterial degradation of hydrocarbons and thus contribute to the biological remediation of contaminated environments. These enzymes have been studied for years in the group of the Marburg microbiology professor Dr. Johann Heider, who in cooperation with Prof. Dr. Maciej Szaleniec from the Jerzy Haber Institute of Catalysis and Surface Chemistry PAS in Krakow presents new results on their mechanism and biotechnological applicability in the journal "ACS Catalysis". The model system of these enzymes is benzyl succinate synthase (BSS), which stereospecifically synthesizes (R)-benzyl succinate from toluene and fumarate. This enzyme is only active after the conversion of a conserved glycine in the active center to a glycyl radical and in this form is extremely sensitive to inactivation by oxygen. In addition, active BSS could only be obtained in small amounts from a few anaerobic toluene-degrading bacteria, which greatly impedes the availability and handling of the enzyme. In the new manuscript, a method for the recombinant production of activated BSS is presented for the first time, which has already been used to characterize some specifically produced mutants. The work further confirms the postulated mechanism of the BSS, which was created from the crystal structure of the non-activated enzyme and mathematical model calculations.
Both substrates, toluene and fumarate, bind in a pocket of the enzyme, while the latter is in the relatively stable glycyl radical form. The fumarate is bound by contacts of the two carboxyl groups with a conserved arginine and with protein backbone atoms, while the toluene is appropriately aligned by hydrophobic amino acids in the wall of the binding pocket. The reaction then proceeds via a radical cascade: first, the glycyl radical abstracts a hydrogen atom from a conserved cysteine in the active center, which generates a highly reactive thiyl radical, which then activates toluene to a benzyl radical. This substrate radical adds to the double bond of fumarate and forms a benzylsuccinyl product radical, which is further converted via a retrograde radical cascade: a hydrogen atom is first removed from the cysteine to generate the product, the radical migrates back to glycine and the product is released from the binding pocket.
The further questions studied in the current work arose from working on the BSS mechanism. For example, two isoleucines of the hydrophobic binding pocket are conserved in most BSS enzymes from different bacteria, but are replaced by valines in two known xylene-converting enzymes. The new study revealed that replacing just one of these isoleucines by a valine led to an enzyme variant that converts m-xylene in addition to toluene. The experimental data were confirmed by molecular dynamic calculations on the computer, which showed a specific expansion of the substrate spectrum for m-xylene. It is particularly noteworthy that the effects of a single additional carbon atom in the substrate are compensated for by the removal of a single carbon atom in the enzyme. Another target for targeted mutagenesis was the fumarate-binding arginine residue, which is universally conserved in all known fumarate-adding enzymes. Nevertheless, it was possible to replace this amino acid with a lysine without losing the activity of the BSS. This mutant also showed a remarkable novel activity, namely the formation of an adduct of toluene and the fumarate analogue acetylacrylate, in which one of the two carboxyl groups is replaced by a keto group. Overall, the results of the study show that BSS (as well as other fumarate-adding enzymes) apparently are not only useful for their physiological reactions, but can also be made to catalyze novel addition reactions between hydrocarbons and olefins with simple changes. This may open up a new way of using these enzymes for various C-C linkages in synthetic approaches.
Professor Dr. Johann Heider teaches microbial biochemistry at the Philipps University Marburg. Professor Maciej Szaleniec has been head of the Joint Laboratory of Biotechnology and Enzyme Catalysis at ICSC PAS since 2010 and is currently Deputy Director for the Research of the ICSC PAS in Krakow. In addition to the working groups of Heider and Szaleniec, Dr. Uwe Linne from the University Marburg and Rainer Meckenstock from the University Duisburg-Essen contributed to the study.
The German Research Foundation and the National Science Center Poland Poland funded the underlying scientific work via the Beethoven Life Grant Program (He2190 / 7-2, He2190 / 13-1, 2018/31 / F / 619 NZ1 / 01856); further support came from PL-Grid (Cyfronet) and the LOEWE-617 Center for Synthetic Microbiology.
Original publication: Salii I., Szaleniec M., Alhaj Zein A., Seyhan D., Sekula A., Schühle K., Kaplieva-Dudek I., Linne U., Meckenstock R. U. & Heider J. (2021) Determinants for substrate recognition in the glycyl radical enzyme benzylsuccinate synthase revealed by targeted mutagenesis. ACS Catal.11, 3361-3370