Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation

Abstract

Metabolite-level regulation of enzyme activity is important for coping with environmental shifts in bacteria. Improved understanding of such regulation could guide attempts to engineer more efficient strains for biochemical production. Recently developed proteomics methods allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feed-back and feed-forward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in four potential metabolic engineering host that fix CO2 using the Calvin cycle, including two photoautotrophs (cyanobacteria) and two chemoautotrophs. Species-specific interactions were observed, such as interactions with glucose-6-phosphate in the chemoautotroph Cupriavidus necator and interactions with glyoxylate in the cyanobacteria Synechocystis sp. PCC 6803, which suggests that metabolite-level regulation could be adapted to a certain metabolic capacity or lifestyle of these bacteria. Identified metabolite interactions with Calvin cycle enzymes fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) and transketolase were tested for effects on catalytic activity using kinetic assays. GAP increased the activity of both Synechocystis and Cupriavidus F/SBPase, which may act as a feed-forward activation mechanism in the Calvin cycle. A kinetic model incorporating regulations on F/SBPase generally enhanced flux control of ATP and NADPH supply over the cycle. We show that LiP-SMap is a promising technique to explore and uncover novel post-translational metabolic regulation, although the method could benefit from improved sensitivity and specificity.