Investigations about two promiscuous aldolases in Nicotiana tabacum

Aldolases (for example fructose-1,6-bP aldolase, transaldolase, 2-keto-3-deoxy-6-phosphogluconate aldolase) are enzymes that catalyze aldol reactions in which carbon-carbon bonds are either split or formed. They are mostly involved in the carbohydrate metabolism like in the CBB cycle, glycolysis, the OPP and ED pathways and gluconeogenesis, but also in glyoxylate and amino acid (mostly arginine and proline) metabolism. Aldolases are often promiscuous, and some have moonlighting functions. In this study, two aldolases (XP 016444199 and XP 016461758) from Nicotiana tabacum were investigated. At the beginning of the study, it was assumed that both enzymes were Entner-Doudoroff aldolases (EDA), which are key enzymes that split 2-keto-3-deoxy-6-phosphogluconate (KDPG) as the second reaction of the classical ED pathway. This assumption was based on in vitro assays with a homolog aldolase from the plant Hordeum vulgare, that was identified as a KDPG aldolase (Chen et al., 2016). However, during the course of this thesis it became evident that the ED pathway does neither exist in plants nor in cyanobacteria as the first enzyme of this pathway, the assumed Entner-Doudoroff dehydratase (EDD), was misinterpreted and is instead a dihydroxy-acid dehydratase (DHAD), which is involved in the synthesis of branched chain amino acids (Evans et al., 2024). Based on this finding, the function of the KDPG aldolases (XP 016444199 and XP 016461758) from Nicotiana tabacum, which will be referred to as NtEDA, was completely unknown. The aim of this thesis was to find clues to elucidate the function of these aldolases. Several eda deletion mutant lines were generated. Growth and the resumption of a dark arrested CBB cycle were not compromised in these putative mutants. Experiments with an antibody that was raised against NtEda from tobacco suggested that the protein may still be expressed in the putative eda mutants despite the deletions that were detected at the genetic level. It is discussed whether this could be the reason for the lack of differences between the presumed NtEDA deletion mutants and the tobacco wildtype, it however remained ultimately uncertain if the antibody was specific against NtEDA. Transcription analyses revealed that EDA is transcribed in seedlings, and in the roots, stems, leaves, and flowers of older Nicotiana tabacum plants and is therefore found in organs with both photoautotrophic and heterotrophic metabolism. In protoplasts EDA was exclusively localized in the chloroplasts in tobacco, barley and rice and was found in chloroplasts and the cytosol in maize. In soybean, GmEDA was also observed in chloroplasts (Evans et al., 2024). The subcellular localization supports the assumption that the EDA aldolases are involved in the CBB cycle and glycolytic routes. Immunoblots with protein extracts from isolated chloroplasts and the described NtEda antibody indicated that the putative NtEDA and HvEDA are located in the soluble and not the membrane fraction of chloroplasts in tobacco and barley. In the cyanobacterium Synechocystis sp. PCC 6803, SynEDA was tagged with a polyhistidine tag (His- tag) and was subsequently detected with a respective antibody against the His-tag in the soluble fraction of the cell. Taken together these data provide first evidence that the investigated aldolases are most likely promiscuous, are transcribed in organs with photoautotrophic and heterotrophic metabolism, and are predominantly localized in the chloroplasts. Their involvement in the carbohydrate metabolism of photoautotrophs is discussed.