**8. Thiamin and Chloroplast Interactions**

In the chloroplast, TPP-dependent enzymes play a role in photosynthesis (α-ketose transketolase, TK, and 1-deoxy-D-xylulose-5-phosphate synthase, DXPS), pentose phosphate pathway (TK), and branched amino acid synthesis (acetohydroxyacid synthase, AHAS). The primary pathway for carbon fixation in plants is the Calvin–Benson cycle (also known as the C3 cycle), which is connected to several other pathways via its intermediates. TK has a central location in the Calvin–Benson cycle catalyzing the reversible transfer of a molecule with two carbons from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate (G3P), generating xylulose 5-phosphate (Xu5P) and ribose 5-phosphate, or from fructose 6-phosphate to produce Xu5P and erythrose 4-phosphate. These reactions are essential for the regeneration of ribulose 1,5-bisphosphate in the Calvin–Benson cycle maintaining active photosynthetic rates and providing precursor molecules for the shikimic acid pathway and phenylpropanoid metabolism (erythrose 4-phosphate) [122].

Additionally, TK is the main TPP-dependent enzyme in the pentose phosphate pathway. Different from the Calvin–Benson cycle, which utilizes CO2, NADPH, and ATP to produce hexose sugars, the pentose phosphate pathway utilizes hexose substrates to produce NADPH and pentoses while releasing CO2. Through participation in the pentose phosphate pathway, transketolase has three important functions in the metabolism of the cells: (i) provision of pentoses for the synthesis of nucleotides; (ii) provision of metabolites for glycolysis or gluconeogenesis pathways; and (iii) indirectly influencing the synthesis of NADPH, required for the anabolic processes and antioxidants reduction (glutathione, ascorbate). Consequently, an appropriate activity of transketolase is essential for the proper functioning of lipid and carbohydrate metabolism [27].

Chlorophyll and carotenoids are synthesized in the methylerythritol pathway (MEP) and the first reaction of this pathway is catalyzed by DXPS; this enzyme combines G3P from the Calvin–Benson cycle with pyruvate from the glycolytic pathway to form deoxyxylulose 5-phosphate (DXP) [26]. Additionally, DXPS is a TPP-dependent enzyme and the product of the DXPS reaction, DXP, is the first substrate in the biosynthesis of the hydroxyethylthiazole phosphate (HETP) moiety of the thiamin molecule itself, and thus TPP [123].

The acetohydroxyacid synthase (AHAS, also known as acetolactate synthase, ALS) is also a TPP-dependent enzyme. It catalyzes the first reaction in the synthesis of the branched-chain amino acids, valine, leucine, and isoleucine, which are only produced in plants [124]. This enzyme is responsible for converting two molecules of pyruvate into 2-acetolactate, the first reaction in a three-step pathway used to produce the three amino acids [124].

## **9. Thiamin and Mitochondria Interactions**

Thiamin participates in the mitochondria central metabolism by functioning as a cofactor forpyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (OGDH), and branched chain 2-oxoacid dehydrogenase (BCOADH, or branched chain ketoacid dehydrogenase, BCKDH). The PDH complex has a central role in bioenergetic processes, controlling the supply of acetyl-CoA into the TCA cycle and anabolic reactions, linking glycolysis and the TCA cycle via the oxidative decarboxylation of pyruvate [27,125]. PDH also produces acetyl-CoA from pyruvate in the chloroplast, which is used in the synthesis of fatty acids [27].

In the TCA cycle itself, OGDH catalyzes a rate-limiting step [27,125]. It converts 2-oxoglutarate, coenzyme A, and NAD<sup>+</sup> to succinic acid, while releasing NADH and CO2 as part of the process. It has also been shown in several studies to be a key regulation point in plant metabolism [126–128]. Additionally, it is responsible for the distribution of succinyl-CoA and 2-oxoglutarate for substrate level phosphorylation of GDP, ADP, or for the synthesis of several amino acids and heme group [27,129].

Another TPP-dependent enzyme, BCOADH, catalyzes the breakdown of the branched chain amino acids, and studies have shown its important role in amino acid metabolism in Arabidopsis [130]. In addition, the enzyme pyruvate decarboxylase (PDC) in the cytosol also requires thiamin as a cofactor. It functions to break down pyruvate to acetaldehyde, an essential step for energy production via alcoholic fermentation under anoxia in Arabidopsis [131,132].
