Exploring Fluorinase Substrate Tolerance at C-2 of SAM

Area of Science:

• Biochemistry
• Enzymology
• Medicinal Chemistry

Background:

• Fluorinase (EC 2.5.1.63) catalyzes the conversion of fluoride and S-adenosyl-L-methionine (SAM) into 5'-fluoro-5'-deoxy-adenosine (5'-FDA) and L-methionine (L-Met).
• The enzyme exhibits strict substrate specificity but shows some tolerance for modifications at the C-2 position of the adenine ring of SAM.

Purpose of the Study:

• To investigate the substrate tolerance of fluorinase with various substituents at the C-2 position of the adenine ring of SAM.
• To identify new fluorinase activities and understand the structural basis for substrate selectivity.
• To assess the potential of fluorinase for developing fluorine-18 radiolabeling agents for Positron Emission Tomography (PET).

Main Methods:

• Enzymatic assays were performed using modified SAM substrates with -NHR, -N3, -OR, and -SR substituents at the C-2 position.
• In silico analysis, including molecular modeling, was employed to investigate enzyme-substrate interactions.
• The study evaluated the catalytic activity and substrate specificity of fluorinase against these novel substrates.

Main Results:

• Fluorinase demonstrated new catalytic activities with NH-methyl, NH-propyl, NH-butyl, and O-butyl substituted SAM analogs.
• Azide (-N3) and thioether (-SR) substituents at the C-2 position were not tolerated by the enzyme.
• In silico analysis revealed favorable hydrogen bonding interactions between NH and O substituents and active site residues (N278 and A279 backbone amide).

Conclusions:

• The fluorinase enzyme exhibits specific substrate preferences, tolerating certain modifications at the adenine C-2 position.
• Understanding these interactions enhances the enzyme's utility as a tool for selective radiolabeling.
• This research supports the development of fluorine-18 labeled compounds for PET imaging applications.