Noncanonical Radical SAM Enzyme Chemistry Learned from Diphthamide Biosynthesis

Area of Science:

• Biochemistry
• Enzymology
• Radical Chemistry

Background:

• Radical S-adenosylmethionine (SAM) enzymes catalyze challenging reactions using SAM and [4Fe-4S] clusters.
• A noncanonical type of radical SAM enzyme is involved in diphthamide biosynthesis, modifying translation elongation factor 2.

Purpose of the Study:

• To elucidate the mechanism of noncanonical radical SAM enzymes in diphthamide biosynthesis.
• To understand the distinct regioselective cleavage of SAM observed in these enzymes.

Main Methods:

• Investigating the binding of SAM to diphthamide biosynthetic radical SAM enzymes.
• Analyzing the role of the [4Fe-4S] cluster in SAM cleavage and radical generation.

Main Results:

• Diphthamide biosynthetic radical SAM enzymes bind SAM with a unique geometry.
• A specific iron in the [4Fe-4S] cluster attacks the ACP group of SAM, forming an organometallic intermediate.
• Homolysis of this intermediate releases the ACP radical, essential for EF2 modification.

Conclusions:

• The distinct SAM binding geometry enables regioselective C-S bond cleavage in noncanonical radical SAM enzymes.
• This mechanism generates a 3-amino-3-carboxypropyl (ACP) radical, differing from the 5'-deoxyadenosyl radical of canonical enzymes.
• Understanding this pathway provides insights into enzyme catalysis and protein modification.