Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor

Ma, Baixu1; Britt, R. David2; Tao, Lizhi1

2024-03-01

10.1021/jacs.3c11266

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   影响因子和分区

0002-7863

1520-5126

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 epsilon-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-N epsilon-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.

SCI ; EI

英语

第一 ; 通讯

National Institute of General Medical Sciences[20231120094247002] ; Shenzhen Science and Technology Program[92353301] ; National Natural Science Foundation of China[1R35GM126961-01]

Chemistry

Chemistry, Multidisciplinary

WOS:001179262800001

AMER CHEMICAL SOC

Web of Science

1.Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China
2.Univ Calif Davis, Dept Chem, Davis, CA 95616 USA

Ma, Baixu,Britt, R. David,Tao, Lizhi. Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor[J]. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY,2024,146(10).

Ma, Baixu,Britt, R. David,&Tao, Lizhi.(2024).Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor.JOURNAL OF THE AMERICAN CHEMICAL SOCIETY,146(10).

Ma, Baixu,et al."Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor".JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 146.10(2024).