**3. The biochemistry behind scurvy**

In the search for a common origin of at least some of the many symptoms observed in scurvy, collagen-related disorders caught the eye of early investigators. Fractures, bleeding, and poor wound healing, all relate to the malfunctioning of tissues (bones, blood vessels, skin) containing a substantial amount of different collagen forms [12, 13]. One striking feature of collagens is the presence in the polypeptide chain of hydroxyproline (hypro), and hydroxylysine, two unusual amino acids which are not incorporated as such during the protein synthesis process, but result from the post-translational modification of selected proline and lysine residues catalyzed by the enzymes prolyl-4-hydroxylase (P4H) and lysyl hydroxylase [14, 15]. Hydroxylated residues have an essential role in collagen stabilization, and underhydroxylated collagen is not functional at all. Collagen underhydroxylation was observed in scurvy animals, a condition reversed by AsA administration [16]. Eventually, AsA involvement in the reaction catalyzed by P4H was ascertained [17, 18]. Still, some doubts occurred about the actual role of AsA in P4H-mediated catalysis. It should be considered that 2-ODDs share a complex catalytic mechanism requiring, besides a specific substrate, molecular oxygen, 2-oxoglutarate, Fe2+ and AsA [19]. Some studies suggested that AsA is required to keep iron in the reduced state [20], implying that any reducing agent able to reduce Fe3+ to Fe2+ could replace AsA, but this opinion is contradicted by the high specificity of AsA as the anti-scurvy factor *in vivo*.

Although AsA involvement in the reactions catalyzed by collagen hydroxylases explains many scurvy symptoms, some others (fatigue, neurological symptoms, impaired vision) are definitely not related to collagen malfunctions. This issue remained unsolved until it became clear that collagen hydroxylases share their catalytic mechanism with a vast array of enzymes, within the large class of 2-oxoglutarate-dependent dioxygenases (2-ODDs) [21]. Checking the long list of 2-ODDs, and comparing it with scurvy symptoms, potential candidates can be spotted, whose inactivation is likely to be caused by AsA deficiency, in analogy to what is observed with collagen hydroxylases. As an example, fatigue and lethargy are the consequences of impaired energy metabolism, possibly caused by impaired activity of two enzymes involved in carnitine biosynthesis: ε-N-trimethyllysine hydroxylase and γ-butyrobetaine hydroxylase [22]. Indeed, AsA deficiency affects carnitine synthesis [23]. It is reasonable to assume that also the remaining scurvy symptoms are somehow related to the inactivation of the other 2-ODDs. A partial list of enzymes using AsA for their catalytic activity is reported in the EBI (European Bioinformatics Institute) CoFactor database [24]. Eighteen out of the 20 enzymes reported, belong to the 2-ODD class.
