Cofactor Generation Cascade for α-Ketoglutarate and Fe(II)-Dependent Dioxygenases

Abstract

Fe(II)- and α-ketoglutarate dependent dioxygenases have emerged as important catalysts for the preparation of non-natural amino acids. The stoichiometric supply of the cosubstrate α-ketoglutarate (αKG) is an important cost factor. A combination of the N -succinyl amino acid hydroxylase SadA with an l -glutamate oxidase (LGOX) allowed for coupling in situ production of αKG to stereoselective αKG-dependent dioxygenases in a one-pot/two-step cascade reaction. Both enzymes were used as immobilized enzymes and tested in a preparative scale setup under process-near conditions. Oxygen supply, enzyme, and substrate loading of the oxidation of glutamate were investigated under controlled reaction conditions on a small scale before upscaling to a 1 L stirred tank reactor. LGOX was applied with a substrate concentration of 73.6 g/L (339 mM) and reached a space-time yield of 14.2 g/L/h. Additionally, the enzyme was recycled up to 3 times. The hydroxylase SadA reached a space-time yield of 1.2 g/L/h at a product concentration of 9.3 g/L (40 mM). For both cascade reactions, the supply with oxygen was identified as a critical parameter. The results underline the robustness and suitability of α-ketoglutarate dependent dioxygenases for application outside of living cells.

Overview of Amino Acid Modifications by Iron- and α-Ketoglutarate-Dependent Enzymes

“…In addition to PCP-bound substrates, occasionally N -acylated substrates are hydroxylated by AAH enzymes en route to free OH-AAs (Figure B). For example, SadA is an AAH from Burkholderia ambifaria AMMD, which acts on numerous N -succinyl- l -AAs (e.g., l -Leu, l -Val, l -Ile, l -Cys, l -Met, l -Ala, l -Phe, l -Asp, l -Glu) with various levels of activity . The desuccinylase LasA then unmasks the α-NH 2 to form 3 R -OH-L-AA products.…”

Oxygenating Biocatalysts for Hydroxyl Functionalisation in Drug Discovery and Development

“…[122] Kourist et al have developed the SadA system by coupling the Fe/αKG with L-glutamate oxidase (LGOX), for the in situ production of co-substrate αKG from L-glutamate. [123] Zaparucha et al have used a genome-mining approach to identify Fe/αKGs able to perform hydroxyl functionalisation with high regio-and stereoselectivity on basic amino acids L-lysine, L-arginine and L-ornithine. [124,125] Kino et al have studied a novel Fe/αKG from Sulfobacillus thermotolerans Y0017 and shown its ability to catalyse the regio-and stereoselective threo-β-hydroxylation of L-histidine and L-glutamine on a preparative scale.…”

Discovery of New Fe(II)/α-Ketoglutarate-Dependent Dioxygenases for Oxidation of l-Proline

“…coli strain lacking the isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase was designed, channeling the endogenously produced cosubstrate α-ketoglutarate toward the αKGDs. In addition, in a one-pot enzymatic cascade reaction with the αKGD N -succinyl amino acid hydroxylase (SadA) catalyzing the hydroxylation of N -succinyl- l -valine to N -succinyl-β-hydroxy- l -valine, in situ production of αKG from l -glutamic acid was resolved with l -glutamate oxidase . In the absence of these approaches, αKGDs can still be considered cost-effective since the reaction requires cheap and abundant O 2 and αKG, which is less expensive than NAD­(P)­H.…”

“…In addition, in a one-pot enzymatic cascade reaction with the αKGD N-succinyl amino acid hydroxylase (SadA) catalyzing the hydroxylation of N- succinyl-L-valine to N-succinyl-β-hydroxy-L-valine, in situ production of αKG from L-glutamic acid was resolved with L-glutamate oxidase. 26 In the absence of these approaches, αKGDs can still be considered cost-effective since the reaction requires cheap and abundant O 2 and αKG, which is less expensive than NAD(P)H. Furthermore, in contrast to P450s, αKGDs are self-sufficient enzymes and do not require a dedicated reductase partner. 27 As an alternative to enzyme engineering, we have chosen to perform genome mining for novel enzymes.…”