Biochemistry and structural studies of kynurenine 3‐monooxygenase reveal allosteric inhibition by Ro 61‐8048

Abstract

The human kynurenine 3-monooxygenase (hKMO) is a potential therapeutic target for neurodegenerative and neurologic disorders. Inhibition of KMO by Ro 61-8048, a potent, selective, and the most widely used inhibitor of KMO, was shown effective in various models of neurodegenerative or neurologic disorders. However, the molecular basis of hKMO inhibition by Ro 61-8048 is not clearly understood. Here, we report biochemistry studies on hKMO and crystal structures of an hKMO homolog, pfKMO from Pseudomonas fluorescens, in complex with the substrate l-kynurenine and Ro 61-8048. We found that the C-terminal ∼110 aa are essential for the enzymatic activity of hKMO and the homologous C-terminal region of pfKMO folds into a distinct, all-α-helical domain, which associates with the N-terminal catalytic domain to form a unique tunnel in proximity to the substrate-binding pocket. The tunnel binds the Ro 61-8048 molecule, which fills most of the tunnel, and Ro 61-8048 is hydrogen bonded with several completely conserved residues, including an essential catalytic residue. Modification of Ro 61-8048 and biochemical studies of the modified Ro 61-8048 derivatives suggested that Ro 61-8048 inhibits the enzyme in an allosteric manner by affecting the conformation of the essential catalytic residue and by blocking entry of the substrate or product release. The unique binding sites distinguish Ro 61-8048 as a noncompetitive and highly selective inhibitor from other competitive inhibitors, which should facilitate further optimization of Ro 61-8048 and the development of new inhibitory drugs to hKMO.-Gao, J., Yao, L., Xia, T., Liao, X., Zhu, D., Xiang, Y. Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

Ommochromes in invertebrates: biochemistry and cell biology

“…Interestingly, KMO localises at the outer mitochondrial membrane, which links this enzyme and the ommochrome pathway to the oxidative metabolism. To date, no crystal structure of any insect KMO exists; only yeast and Pseudomonas KMO have been successfully purified and crystallised (Amaral et al, ; Smith et al, ; Gao et al, ). New KMO inhibitors based on crystallographic data could provide tools to study the ommochrome pathway in model and non‐model organisms.…”

Kynurenine‐3‐monooxygenase: A new direction for the treatment in different diseases

“…Studies on the structure and mechanism of KMO in animal (Zhang et al, ) have been discussed in the past (Kim et al, ; Smith et al, ). KMO is situated in the outer membrane of mitochondria (Quan et al, ) as a membrane‐associated protein (Gao et al, ), and its crystal structure was found by Amaral et al () as well as the first successful bacterial ( Escherichia coli ) expression of active human KMO enzyme expressed in the soluble fraction found by Wilson et al (). A relative gauge of KMO activity by using mass spectrometry‐multiple‐reaction monitoring (MS‐MRM) (Winkler et al, ) and RapidFire mass spectrometry (RF‐MS) (Lowe et al, ) was detected and a more exact and effective screening of this class of enzymes in multiple assay formats was permitted.…”

Targeting tryptophan catabolic kynurenine pathway enhances antitumor immunity and cytotoxicity in multiple myeloma

“…Functional-grade PD-L1 blocking antibody (antihuman PD-L1, clone MIH1) was obtained from eBiosciences [4]. Ro 61-8048 [13] and INCB 024360 were purchased from Selleck Chemicals. WST-1 Cell Proliferation Reagent was purchased from Clontech Laboratories, Inc. (USA).…”

“…We therefore next assessed whether KMO inhibition affects maturation and immune function of pDCs in MM. For these studies, we treated pDCs from MM patients with nontoxic concentrations of a specific inhibitor of KMO Ro 61-8048 (0.1-0.2 µM) [13], and examined changes in activation/ maturation markers on pDCs. Ro-61-8048 triggers upregulation of CD83 and CD80 on pDCs (Fig.…”