, 2011). To perform a more thorough analysis, we analysed purified products of the l-leucine hydroxylation catalysed by IDO, MFL and GOX. We chose MFL and GOX because of the effectiveness with which l-leucine hydroxylation was catalysed by these enzymes (see Table 2). Both LC/ESI-MS and 1H-NMR analyses showed that all compounds examined were identical to 4-hydroxyleucine (Fig. 3b).

A similar investigation showed that all enzymes examined oxidize l-methionine to l-methionine sulfoxide. For products of l-threonine hydroxylation, we performed ESI-MS (m/z = +166.1 [M+H], estimated molecular mass 165.21), the results of which indicated that the purified compound was hydroxylated l-threonine. Based on the l-threonine molecular

structure, and in the light of the preference of IDO homologues to hydroxylate at the C4 position, we concluded that BPE and AVI hydroxylate l-threonine to 4-hydroxythreonine (Fig. 3c). 3-Methyladenine ic50 Therefore, the PF10014 members from the first, second and third groups have threefold substrate specificities. These enzymes oxidize l-methionine and hydroxylate l-Leucine, but the third substrate could be variable. For example, l-isoleucine is a substrate of enzymes from the first and second groups, but l-threonine is a substrate for those from the third group. Two enzymes from the sixth group (GOX and MFL) have dual l-methionine/l-leucine substrate specificity. The GVI (sixth group) and PLU (fifth group) dioxygenases could only oxidize l-methionine and were excluded from further analysis because of their low activities and high KM values (data not shown). Perhaps, Cabozantinib these enzymes have evolutionary adapted to hydroxylate-specific substrates other than free l-amino acids. An investigation of the kinetic parameters of dioxygenase hydroxylation of free l-amino acids suggested the existence of a novel dioxygenase subfamily within the PF10014 family (Table 2). This novel subfamily can be characterized as a subset of enzymes

for which free l-amino acids could Suplatast tosilate be accepted as in vivo substrates. This family has at least three apparent ‘evolutionary attractors’ of their substrate specificity: l-isoleucine (IDO), l-threonine (BPE) and l-leucine (GOX, MFL). In each case, the corresponding dioxygenases have ‘physiological’ KM values for these l-amino acids (Table 2). Previously, we proposed that the hydroxylation of l-isoleucine, coupled with the oxidation of α-ketoglutarate to succinate, serves as a tricarboxylic acid cycle (TCA) shunt to compensate for the absence of α-ketoglutarate dehydrogenase activity in B. thuringiensis (Smirnov et al., 2010; Ogawa et al., 2011). From this standpoint, hydroxylated l-isoleucine is the only byproduct of succinate synthesis; however, this hypothesis remains unproven. As mentioned previously, the induction of IDO, AR and RhtA in B.