Entner-Doudoroff pathway in Synechocystis PCC 6803: Proposed regulatory roles and enzyme multifunctionalities

Abstract

The Entner-Doudoroff pathway (ED-P) was established in 2016 as the fourth glycolytic pathway in Synechocystis sp. PCC 6803. ED-P consists of two reactions, the first catalyzed by 6-phosphogluconate dehydratase (EDD), the second by keto3-deoxygluconate-6-phosphate aldolase/4-hydroxy-2-oxoglutarate aldolase (EDA). ED-P was previously concluded to be a widespread (∼92%) pathway among cyanobacteria, but current bioinformatic analysis estimated the occurrence of ED-P to be either scarce (∼1%) or uncommon (∼47%), depending if dihydroxy-acid dehydratase (ilvD) also functions as EDD (currently assumed). Thus, the biochemical characterization of ilvD is a task pending to resolve this uncertainty. Next, we have provided new insights into several single and double glycolytic mutants based on kinetic model of central carbon metabolism of Synechocystis. The model predicted that silencing 6-phosphogluconate dehydrogenase (gnd) could be coupled with ∼90% down-regulation of G6P-dehydrogenase, also limiting the metabolic flux via ED-P. Furthermore, our metabolic flux estimation implied that growth impairment linked to silenced EDA under mixotrophic conditions is not caused by diminished carbon flux via ED-P but rather by a missing mechanism related to the role of EDA in metabolism. We proposed two possible, mutually non-exclusive explanations: (i) Δeda leads to disrupted carbon catabolite repression, regulated by 2-keto3-deoxygluconate-6-phosphate (ED-P intermediate), and (ii) EDA catalyzes the interconversion between glyoxylate and 4-hydroxy-2-oxoglutarate + pyruvate in the proximity of TCA cycle, possibly effecting the levels of 2-oxoglutarate under Δeda. We have also proposed a new pathway from EDA toward proline, which could explain the proline accumulation under Δeda. In addition, the presented in silico method provides an alternative to 13C metabolic flux analysis for marginal metabolic pathways around/below the threshold of ultrasensitive LC-MS. Finally, our in silico analysis provided alternative explanations for the role of ED-P in Synechocystis while identifying some severe uncertainties.

Dark respiration modulates photosynthetic carbon yield in cyanobacteria

“…Our results thus strongly support that the ED pathway is incomplete in S. elongatus with the KDPG aldolase participating in respiration through its alternative decarboxylation activity. This is also consistent with the findings that the KDPG aldolase is much more prevalent than 6PGDH in cyanobacteria 18 .…”

“…6). A recent bioinformatics study analyzed the possible cooccurrence of four glycolytic pathways (EMP, ED, OPP, and phosphoketolase) in cyanobacteria and found that the phosphoketolase and OPP pathways had the highest co-occurrence probability of 84.8% compared to all other combinations 18 , supporting the OPP-phosphoketolase route in the dark during cyanobacterial diel light/dark growth. In addition, phosphoketolase exists in other eukaryotes such as diatom (Supplementary Fig.…”

“…Two key enzymes of the ED pathway are the 6-phosphogluconate (6PG) dehydratase (6PGDH) and the KDPG aldolase. However, no genuine 6PGDH gene has been annotated in the S. elongatus genome, and rarely exists in other cyanobacteria 18 . Previous research suggests that dihydroxy acid dehydratase (DHAD) ( ilvD ) in amino acid metabolism could serve as an alternative enzyme for the dehydratase activity in the ED pathway in cyanobacteria 17 .…”

Plastid ancestors lacked a complete Entner-Doudoroff pathway, limiting plants to glycolysis and the pentose phosphate pathway

“…Early metabolic studies in Entamoeba histolytica 34 , Aspergillus niger 35 , and Penicillium notaturn 36 suggested it was also a significant metabolic route in unicellular or filamentous eukaryotes (reviewed in 2 ). More recently, the enzymatically similar DHAD class of dehydratases from the BCAA pathway has been implicated in the ED pathway, leading to speculation that the ED pathway operates in plants and diatoms [11][12][13] . Based on biochemical characterization of DHAD and EDD substrate specificity (Fig.…”

“…Due to their common evolutionary origin and high degree of amino acid similarity, DHADs and EDDs have been treated collectively in phylogenetic analyses aimed at understanding the natural distribution of the ED pathway 11 . The assumption that they have overlapping substrate specificities and biochemical functions 12 has led to the conclusion that a functional ED pathway operates in flowering plants, mosses, ferns 11 , and diatoms 13 in addition to prokaryotes 4 . Bacteria possess both types of dehydratase, but in plants a definitive physiological role has thus far only been established for DHAD 14 .…”