Affinity-peptide-mediated multi-enzyme self-assembly system enhances dioxygenase catalyzing C–H hydroxylation via in situ α-ketoglutarate generation and H2O2 elimination

Fe(II)/α-ketoglutarate-dependent dioxygenases (Fe(II)/α-KG DOs) are powerful biocatalysts for CH bond hydroxylation, generating diverse high-value molecules. An inadequate cosubstrate α-KG supply leads to low yields of the desired products. Currently, L-glutamate oxidase (LGOX) is used to generate α-KG and cascades with catalase (KatG) to eliminate H2O2 (a by-product of LGOX), an inhibitor of Fe(II)/α-KG DOs. However, free enzymes still cause H2O2 accumulation, inhibiting Fe(II)/α-KG DO activity. Here, we employed RIAD-RIDD short peptides to form LGOX-KatG specific 1:2 ratio self-assembled tri-enzyme units using a scaffold-free strategy to obtain structural insights via computational analysis and to avoid H2O2 toxicity to achieve high yield of a promising anti-diabetic drug (2S,3R,4S)-4-hydroxyisoleucine in a one-pot system. A conversion of >95 % was achieved at a 100 mM isoleucine within 7 h, with 2.00 g·L−1·h−1 space-time yield. This work provides a universal, industrially attractive approach for supplying a cosubstrate for Fe(II)/α-KG DO catalyzing hydroxylation.