Abstract
For nearly half a century, it was believed that cyanobacteria had an incomplete tricarboxylic acid (TCA) cycle, because 2-oxoglutarate dehydrogenase (2-OGDH) was missing. Recently, a bypass route via succinic semialdehyde (SSA), which utilizes 2-oxoglutarate decarboxylase (OgdA) and succinic semialdehyde dehydrogenase (SsaD) to convert 2-oxoglutarate (2-OG) into succinate, was identified, thus completing the TCA cycle in most cyanobacteria. In addition to the recently characterized glyoxylate shunt that occurs in a few of cyanobacteria, the existence of a third variant of the TCA cycle connecting these metabolites, the γ-aminobutyric acid (GABA) shunt, was considered to be ambiguous because the GABA aminotransferase is missing in many cyanobacteria. In this study we isolated and biochemically characterized the enzymes of the GABA shunt. We show that N-acetylornithine aminotransferase (ArgD) can function as a GABA aminotransferase and that, together with glutamate decarboxylase (GadA), it can complete a functional GABA shunt. To prove the connectivity between the OgdA/SsaD bypass and the GABA shunt, the gadA gene from Synechocystis sp. PCC 6803 was heterologously expressed in Synechococcus sp. PCC 7002, which naturally lacks this enzyme. Metabolite profiling of seven Synechococcus sp. PCC 7002 mutant strains related to these two routes to succinate were investigated and proved the functional connectivity. Metabolite profiling also indicated that, compared to the OgdA/SsaD shunt, the GABA shunt was less efficient in converting 2-OG to SSA in Synechococcus sp. PCC 7002. The metabolic profiling study of these two TCA cycle variants provides new insights into carbon metabolism as well as evolution of the TCA cycle in cyanobacteria.
Highlights
As mankind strives to lower anthropogenic carbon dioxide emissions, our ability to manipulate photosynthetic primary production is critical
Our findings demonstrate the significant involvement of the OgdA/succinic semialdehyde dehydrogenase (SsaD) bypass under dark aerobic growth conditions in Synechococcus 7002, but suggest that there is much lower activity under photoautotrophic conditions
Deconvolution of the MS/MS spectra of succinic semialdehyde (SSA)-DNPH showed an intense fragment at m/z −182.1, which corresponds to the loss of the DNPH group from SSADNPH
Summary
As mankind strives to lower anthropogenic carbon dioxide emissions, our ability to manipulate photosynthetic primary production is critical. Cyanobacteria were recently shown to have a complete but non-traditional TCA cycle (Zhang and Bryant, 2011) In this variation, 2-oxoglutarate (2-OG) is converted to succinate by 2-oxoglutarate decarboxylase (2-OGDC, OgdA) and succinate semialdehyde dehydrogenase (SSADH, SsaD) (Figure 1). 2-oxoglutarate (2-OG) is converted to succinate by 2-oxoglutarate decarboxylase (2-OGDC, OgdA) and succinate semialdehyde dehydrogenase (SSADH, SsaD) (Figure 1) The presence of this bypass in most cyanobacteria corrected a long-held misconception that these organisms have an incomplete TCA cycle due to the absence of 2-oxoglutarate dehydrogenase (2-OGDH), and provided much useful knowledge illustrating the occurrence and physiological functions of TCA variants in these bacteria (Zhang and Bryant, 2011, 2014). Flux-balance calculations have suggested that minimal flux occurs through the bypass reactions under photoautotrophic growth conditions (Hendry et al, 2016)
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