Recently, the Sharkey lab proposed an alternative pathway around the Calvin-Benson cycle, called the glucose-6-phosphate shunt. This is a futile cycle, consuming ATP and releasing CO2. However, it may address unexplained observations about the Calvin-Benson cycle. To understand the regulation and flux through the glucose-6-phosphate shunt, we have characterized two key enzymes. Phosphoglucoisomerase (PGI) provides the substrate for the pathway and glucose-6-phosphate dehydrogenase (G6PDH) is the first committed step. We found that PGI has a higher Km for glucose 6-phosphate (G6P) compared to fructose 6-phosphate (F6P). This, in combination with maintaining a disequilibrium between F6P and G6P, allows it to act as a one-way valve to partition carbon out of the Calvin-Benson cycle. PGI also acts as a key regulatory enzyme in starch synthesis and degradation. We found that plastidic G6PDH is redox regulated. Reduction catalyzed by thioredoxin causes a decrease in substrate affinity but the enzyme maintains activity in the light. This regulation can be overcome by oxidation by hydrogen peroxide or by an increase in substrate concentration. We also found that the midpoint potential of G6PDH is in a range to allow dynamic regulation in the light. In addition to characterization of key enzymes, we investigated whether the G6P shunt is responsible for respiration in the light (RL). We found that all loss-of-function G6PDH isoform mutants decreased RL. This also allowed us to explain respiration phenotypes in a variety of mutant lines. Overall, we have shown that the G6P shunt is the source of RL and have used biochemical characteristics of key enzymes to understand and predict changes in flux through shunt.
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