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Updated: Aug 27, 2025
FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.
Published on: August 25, 2020
Unravelling interactions between active site residues and DMAP in the initial steps of prenylated flavin
Szymon Żaczek1, Agnieszka Dybala-Defratyka1
1Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Żeromskiego 116, 90-924 Lodz, Poland.
Background:
Prenylated flavin mononucleotide (prFMN) is a recently discovered, heavily modified flavin compound. It is the only known cofactor that enables enzymatic 1,3-dipolar cycloaddition reactions. It is produced by enzymes from the UbiX family, from flavin mononucleotide and either dimethylallyl mono- or diphosphate. prFMN biosynthesis is currently reported to be initiated by protonation of the substrate by Glu140.
Methods:
Computational chemistry methods are applied herein - Constant pH MD, classical MD simulations, and QM cluster optimizations.
Results:
Glu140 competes for a single proton with Lys129 prior to prFMN biosynthesis, but it is the latter that adopted a protonated state. Once the prenyl-FMN adduct is formed, Glu140 occurs in a protonated state far more often, while the occupancy of protonated Lys129 does not change. Lys129, Glu140, and Arg122 seem to play a key role in either stabilizing or protonating DMAP phosphate group within the PaUbiX active site throughout initial steps of prFMN biosynthesis.
Conclusions:
The role of Lys129 in the functioning of PaUbiX is reported for the first time. Glu140 is unlikely to act as a proton donor in prFMN biosynthesis. Instead, Lys129 and Arg122 fulfil this role. Glu140 still plays a role in contributing to hydrogen-bond network. This behavior is most likely conserved throughout the UbiX family due to the structural similarity of the active sites of those proteins.
Significance:
Mechanistic insights into a crucial biochemical process, the biosynthesis of prFMN, are provided. This study, although purely computational, extends and perfectly complements the knowledge obtained in classical laboratory experiments.
Insights
Lysine 129 and Arginine 122, not Glutamate 140, initiate prenylated flavin mononucleotide (prFMN) biosynthesis by protonating the substrate. This computational study reveals key roles for these residues in prFMN production by UbiX enzymes.
Area of Science:
- Biochemistry
- Computational Chemistry
- Enzymology
Background:
- Prenylated flavin mononucleotide (prFMN) is a unique cofactor essential for enzymatic 1,3-dipolar cycloaddition reactions.
- UbiX family enzymes synthesize prFMN from flavin mononucleotide and dimethylallyl pyrophosphate.
- Previous understanding suggested Glu140 initiates prFMN biosynthesis via substrate protonation.
Purpose of the Study:
- To computationally investigate the mechanism of prFMN biosynthesis initiation.
- To elucidate the roles of active site residues, particularly Glu140, Lys129, and Arg122, in PaUbiX.
- To provide mechanistic insights complementing experimental findings on prFMN production.
Main Methods:
- Application of computational chemistry techniques, including Constant pH Molecular Dynamics (MD) and classical MD simulations.
- Utilizing Quantum Mechanics (QM) cluster optimizations for detailed analysis.
- Focus on the PaUbiX enzyme active site and substrate interactions.
Main Results:
- Lys129 is protonated before prFMN biosynthesis, while Glu140 is not.
- Glu140 is protonated more frequently after prFMN adduct formation; Lys129 protonation state remains unchanged.
- Lys129, Glu140, and Arg122 collectively stabilize or protonate the dimethylallyl pyrophosphate substrate within the PaUbiX active site.
Conclusions:
- Lys129 and Arg122, not Glu140, are the likely proton donors initiating prFMN biosynthesis.
- Glu140 contributes to the active site's hydrogen-bond network.
- The observed mechanism is likely conserved across the UbiX enzyme family due to conserved active site structures.
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