posted on 2024-03-01, 14:08authored byBaixu Ma, R. David Britt, Lizhi Tao
Pyrrolysine, the 22nd amino acid encoded by the natural
genetic
code, is essential for methanogenic archaea to catabolize methylamines
into methane. The structure of pyrrolysine consists of a methylated
pyrroline carboxylate that is linked to the ε-amino group of
the l-lysine via an amide bond. The biosynthesis of pyrrolysine
requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes
the first biosynthetic step, the isomerization of l-lysine
into methylornithine. PylC catalyzes an ATP-dependent ligation of
methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed
by PylD via oxidation of the PylC product to form pyrrolysine. While
enzymatic reactions of PylC and PylD have been well characterized
by X-ray crystallography and in vitro studies, mechanistic
understanding of PylB is still relatively limited. Here, we report
the first in vitro activity of PylB to form methylornithine
via the isomerization of l-lysine. We also identify a lysyl
C4 radical intermediate that is trapped, with its electronic structure
and geometric structure well characterized by EPR and ENDOR spectroscopy.
In addition, we demonstrate that SAM functions as a catalytic cofactor
in PylB catalysis rather than canonically as a cosubstrate. This work
provides detailed mechanistic evidence for elucidating the carbon
backbone rearrangement reaction catalyzed by PylB during the biosynthesis
of pyrrolysine.