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

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. Although Kcr has been shown to regulate photosynthesis-related proteins in various photosynthetic organisms, the enzymes responsible for this modification have not been identified in cyanobacteria. Moreover, the molecular mechanism by which crotonylation modulates photosynthesis in cyanobacteria is largely unexplored. 

In this study, combining bioinformatic predictions with enzymatic activity assays, researchers identified cyanobacterial Gcn5-related N-acetyltransferase (cGNAT2) as a crotonyltransferase and 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.

By employing anticrotonyllysine antibody and through label-free quantitative crotonylome analysis, researchers identified 536 Kcr sites targeted by cGNAT2 and 360 Kcr sites regulated by CddA. These modified proteins were widely distributed across metabolic pathways and photosynthetic processes. Several key enzymes were co-regulated by both cGNAT2 and CddA, indicating that the Kcr mediated by these enzymes plays a regulatory role in photosynthesis and energy metabolism. 

Furthermore, researchers 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) 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 the two enzymes modulate the Kcr status of PsaD both in vitro and in vivo.

To explore the regulatory role of Kcr in PsaD function, researchers constructed PsaD site-specific mutants. Phenotypic analyses indicated that the mutant strains exhibited significantly reduced growth rates and photosynthetic efficiency under both normal light and high light stress conditions. The results suggested 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 identified lysine crotonyltransferase cGNAT2 and decrotonylase CddA in cyanobacteria, which provides novel insights into the regulation of substrate proteins by these enzymes. Besides, 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.