2-Ketoglutarate-Generated In Vitro Enzymatic Biosystem Facilitates Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Mediated C-H Bond Oxidation for (2,3,4)-4-Hydroxyisoleucine Synthesis.

Fe(II)/2-ketoglutarate-dependent dioxygenase (Fe(II)/2-KG DO)-mediated hydroxylation is a critical type of C-H bond functionalization for synthesizing hydroxy amino acids used as pharmaceutical raw materials and precursors. However, DO activity requires 2-ketoglutarate (2-KG), lack of which reduces the efficiency of Fe(II)/2-KG DO-mediated hydroxylation. Here, we conducted multi-enzymatic syntheses of hydroxy amino acids. Using (2,3,4)-4-hydroxyisoleucine (4-HIL) as a model product, we coupled regio- and stereo-selective hydroxylation of l-Ile by the dioxygenase IDO with 2-KG generation from readily available l-Glu by l-glutamate oxidase (LGOX) and catalase (CAT). In the one-pot system, HO significantly inhibited IDO activity and elevated Fe concentrations of severely repressed LGOX. A sequential cascade reaction was preferable to a single-step process as CAT in the former system hydrolyzed HO. We obtained 465 mM 4-HIL at 93% yield in the two-step system. Moreover, this process facilitated C-H hydroxylation of several hydrophobic aliphatic amino acids to produce hydroxy amino acids, and C-H sulfoxidation of sulfur-containing l-amino acids to yield l-amino acid sulfoxides. Thus, we constructed an efficient cascade reaction to produce 4-HIL by providing prerequisite 2-KG from cheap and plentiful l-Glu and developed a strategy for creating enzymatic systems catalyzing 2-KG-dependent reactions in sustainable bioprocesses that synthesize other functional compounds.