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

Abstract

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 (2s,3r,4s)-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, H2O2 significantly inhibited IDO activity and elevated Fe2+ concentrations of severely repressed LGOX. A sequential cascade reaction was preferable to a single-step process as CAT in the former system hydrolyzed H2O2. 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.

Designing of an Efficient Whole-Cell Biocatalyst System for Converting L-Lysine Into Cis-3-Hydroxypipecolic Acid

“…Several studies have linked glutamate to the 2-OG conversion reaction ( Wu et al, 2018 ; Sun et al, 2019 ). This reaction also produces H 2 O 2 , which can impair enzymatic activity ( Niu et al, 2014 ; Jing et al, 2020 ). In the TCA cycle, 2-OG is an important intermediate metabolite.…”

Efficient and scalable synthesis of 1,5-diamino-2-hydroxy-pentane from l-lysine via cascade catalysis using engineered Escherichia coli

“…Thus, the amount of α-ketoglutarate acid directly influences the catalytic efficiency of Fe(II)/α-ketoglutarate-dependent dioxygenases. Several studies have constructed chassis strains to generate α-ketoglutarate acid and facilitate Fe(II)/α-ketoglutarate-dependent dioxygenase-mediated C–H bond oxidation [ 35 – 37 ]. Here, the effect of the α-ketoglutarate/lysine ratio on E. coli whole-cell catalysis was evaluated.…”

Molecular Insights into the Regioselectivity of the Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Catalyzed C–H Hydroxylation of Amino Acids

“…Various l -proline hydroxylases have been explored to facilitate hydroxylation of substrates at different positions. − l -Lysine dioxygenase (KDO) has also been well studied for catalyzing hydroxylation. − As an efficient biocatalyst, GlbB can hydroxylate l -lysine with high regioselectivity in the preparation of key dipeptide fragments of griseofulvin and has received more attention regarding catalytic performance and thermostability in recent years. − In addition, PolL enables the sequential hydroxylation of α-amino-δ-carbamoylhydroxyvaleric acid . Besides, there are Fe/2-KG DOs that act on amino acids bound to peptidyl carrier proteins. , According to the established phylogenetic tree, Fe/2-KG DOs that hydroxylate amino acids can be generally divided into three clusters based on the properties of their substrates: (i) those that act on hydroxylated heterocyclic amino acid substrates, (ii) those involved in peptide antibiotic synthesis, and (iii) those involved in the oxidation of aliphatic amino acids.…”