Structures of the holoenzyme TglHI required for 3-thiaglutamate biosynthesis

• Published in: Structure (London) Vol. 31; no. 10; pp. 1220 - 1232.e5
• Main Authors: Zheng, Yanhui, Xu, Xiaoqing, Fu, Xiaoli, Zhou, Xuerong, Dou, Chao, Yu, Yue, Yan, Weizhu, Yang, Jingyuan, Xiao, Minqin, van der Donk, Wilfred A., Zhu, Xiaofeng, Cheng, Wei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 05.10.2023
• Subjects: Holoenzymes - metabolism; Iron - metabolism; mechanism; metalloenzymes; Molecular Conformation; Peptides - chemistry; Protein Processing, Post-Translational; RiPP; TglHI holoenzyme; X-ray; RiPP; TglHI holoenzyme; X-ray; mechanism; metalloenzymes
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2023.08.004

Structural diverse natural products like ribosomally synthesized and posttranslationally modified peptides (RiPPs) display a wide range of biological activities. Currently, the mechanism of an uncommon reaction step during the biosynthesis of 3-thiaglutamate (3-thiaGlu) is poorly understood. The removal of the β-carbon from the Cys in the TglA-Cys peptide catalyzed by the TglHI holoenzyme remains elusive. Here, we present three crystal structures of TglHI complexes with and without bound iron, which reveal that the catalytic pocket is formed by the interaction of TglH–TglI and that its activation is conformation dependent. Biochemical assays suggest a minimum of two iron ions in the active cluster, and we identify the position of a third iron site. Collectively, our study offers insights into the activation and catalysis mechanisms of the non-heme dioxygen-dependent holoenzyme TglHI. Additionally, it highlights the evolutionary and structural conservation in the DUF692 family of biosynthetic enzymes that produce diverse RiPPs. [Display omitted] •Crystal structures of the holoenzyme TglHI required for 3-thiaglutamate biosynthesis•Different conformational states of the apo and Fe-bound forms of TglHI•Activation of the TglHI holoenzyme is regulated by the L2/L4 loops•The DUF692 family is evolutionary and structural conserved Here, Zheng et al. present three crystal structures of TglHI, both with and without iron ions, involved in 3-thiaglutamate biosynthesis. This discovery sheds light on the activation of TglHI through conformational changes and offers insights into the structural evolution within the DUF692 enzyme family.