PNNL

Abstract

Cyanobacteria require ultra-fast metabolic switching to maintain cellular reducing power balance during environmental fluctuations. Glucose-6-phosphate dehydrogenase (G6PDH), which catalyzes the rate-limiting step of the oxidative pentose phosphate pathway (OPPP), provides essential NADPH and metabolic intermediates for biosynthetic processes and redox homeostasis. In cyanobacteria, the unique redox-sensitive protein OpcA acts as a metabolic switch for G6PDH, enabling rapid adjustment of reducing power generation from glycogen catabolism resulting in precise regulation of carbon flux between anabolic and catabolic pathways. While redox states and allosteric activation of OpcA’s cysteine-rich structure are known to regulate G6PDH, the detailed mechanisms of how redox post-translational modifications (PTMs) influence OpcA’s allosteric effects on G6PDH structure, dynamic, and function remain elusive. To address this, we combined PTM-psi computational modelling with experimental redox proteomics to investigate how redox-driven thiol PTMs allow OpcA to modulate the structure and function of G6PDH, using Synechococcus elongatus PCC 7942 as a model system. Redox proteomics captures several significantly changed cysteine residues when cyanobacteria are subjected to dark treatments or circadian shifts. The simulation results of these redox modified cysteine sites demonstrate that thiol PTMs on the cysteine residues near the OpcA-G6PDH interface play a crucial role in allosteric regulation of regions affecting the G6PDH activity, including a potential gate region for substrate ingress and product egress, as well as critical hydrogen bond networks within the active site. These PTMs promote rapid metabolic switching by enhancing G6PDH catalytic activity when OpcA is oxidized. This study provides evidence for novel molecular mechanisms that elucidate the importance of cysteine PTMs of OpcA in modulating G6PDH structure and function in an allosteric manner, demonstrating how PTM-level regulation provides a critical control mechanism that enables cyanobacteria to rapidly adapt to environmental fluctuations through precise metabolic fine-tuning.