Lysine crotonylation (Kcr) is an important type of post-translational modification (PTM). Due to its unique planar four-carbon structure and neutral charge, it significantly influences the interactions between proteins and DNA or other proteins, thereby playing a critical role in biological processes such as gene expression regulation and metabolic pathway modulation.

Cyanobacteria are a type of bacteria capable of performing oxygenic photosynthesis and among the earliest organisms on Earth to perform photosynthesis. Although Kcr has been shown to regulate photosynthesis-related proteins in various photosynthetic organisms, including wheat, rice, and tobacco,theThe regulatory enzymes responsible for this modification have not been systematically identified in cyanobacteria. Moreover, the molecular mechanisms by which crotonylation modulates photosynthesis in cyanobacteria remain largely unexplored. 

Recently, a research group led by Prof. GE Feng from the Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences successfully identified a pair of Kcr regulatory enzymes in cyanobacteria. They also revealed how these enzymes affect photosynthesis by specifically regulating the Kcr status of substrate proteins. The study was published in New Phytologist. 

Through a combination of bioinformatic predictions and enzymatic activity assays, the researchers first identified cyanobacterial Gcn5-related N-acetyltransferase (cGNAT2) as a Cyanobacterial Deacetylase/Depropionylase (CddA) as a decrotonylase. Subsequent molecular docking simulations and fluorescence substrate assays confirmed the substrate specificity and catalytic activity sites of these two enzymes.

Subsequently, the researchers employed anticrotonyllysine antibody combined with label-free quantitative crotonylome analysis to identify 536 Kcr sites targeted by cGNAT2 and 360 Kcr sites regulated by CddA. These modified proteins are widely distributed across metabolic pathways and photosynthetic processes. Several key enzymes are co-regulated by both cGNAT2 and CddA, indicating that the Kcr mediated by this pair of enzymes plays a vital regulatory role in photosynthesis and energy metabolism. 

Further investigations revealed that both cGNAT2 and CddA regulate the Kcr status of Photosystem I subunit II (PsaD), which is a core subunit of photosystem I (PSI) that is essential for light harvesting, electron transport and maintaining PSI stability. In vitro enzymatic assays, biolayer interferometry (BLI) and in vivo immunoprecipitation (IP) experiments confirmed that these two enzymes modulate the Kcr status of PsaD both in vitro and in vivo.

In order to explore the regulatory role of Kcr in PsaD function, the researchers constructed PsaD site-specific mutants. Phenotypic analyses indicate that the mutant strains exhibit significantly reduced growth rates and photosynthetic efficiency under both normal light and high light stress conditions. These results suggest that the dynamic balance of Kcr at K28 and K108 may affect PSI stability, thereby influencing the growth and photosynthetic performance of cyanobacteria.

This study is pioneering in its identification of lysine crotonyltransferase cGNAT2 and decrotonylase CddA in cyanobacteria, thereby providing novel mechanistic insights into the regulation of substrate proteins by these enzymes.  The identification of Kcr regulatory enzymes and the functional network of Kcr provide novel insights into the molecular mechanisms of photosynthesis in Syn7002 and possibly in other photosynthetic organisms.

(Editor: MA Yun)