The first committed step for the excretion of bilirubin is reduction of four vinylic bonds to form urobilinogen. This reaction is catalyzed by bilirubin reductase (BilR) that uses four NADHs to accomplish the transformation. Due to the asymmetry of bilirubin, BilR must either recognize 15 different oxidant substrates or have a preferred reduction order. AlphaFold structural prediction indicates structural homology with bacterial 2,4-dienoyl-CoA reductase and 2-napthoyl-CoA reductase, both of which are single-subunit proteins with a two-domain structure that houses three cofactors: an FAD, an Fe4S4 cluster, and an FMN. In this study, anaerobic kinetic and mechanistic analysis of bilirubin reductase from Mediterraneibacter gnavus (MgBilR) is presented. Anaerobic solvent exchange studies define the stereochemistry of hydride transfer from NADH as ProS. Transient-state kinetics of the reductive half-reaction demonstrate that MgBilR can become reduced by four electrons with either NADH or NADPH, but that NADH is the preferred reductant by four orders of magnitude (based on kred/Kd values). Only the first hydride transfer from NADH occurs at a rate that is catalytically relevant. Single-turnover experiments show two dominant phases corresponding to two-electron flavin reduction and subsequent rate-limiting bilirubin reduction that occurs with flavin reoxidation. Single-turnover data correlated with steady-state assays and NMR tracking of the steady-state reaction confirm that BilR can reduce both types of vinylic bonds of bilirubin but appears to have a 4-fold preference for reduction of α-vinylic over enamine bonds that is defined almost entirely by the rate of hydride transfers to the oxidant.
Corine O Smith, Graham R. Moran· Biochemistry· 0 citations
Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that has been implicated in several cancers due to the essential role in the synthesis of glutamine and proline. Inhibition of hOAT may provide a novel method to treat a variety of cancers. In recent years, selective inhibition of hOAT through mechanism-based inactivation has shown promise in the treatment of hepatocellular carcinoma. Inspired by the recently reported hOAT-selective δ-deprotonation and previously proposed ring-strain mediated mechanisms of inactivation, we designed, synthesized, and evaluated (1S,3R,5R)-3-amino-6,6-difluorobicyclo[3.1.0]hexane-1-carboxylic acid (5), a time-dependent inhibitor of hOAT. Structural and mechanistic studies validated the proposed mechanism of 5, which undergoes a ring-opening mechanism to form a PLP-inactivator adduct that is tightly bound in the active site of hOAT. Intact protein mass spectrometry, 19F NMR spectroscopy, transient-state kinetic studies, X-ray crystallography, and QM calculations were used to determine the final adduct and explore the mechanism of inactivation of 5. This is the first report of a ring-opening event in the active site of hOAT.
Allison N Devitt, Abigail L. Vargas, C. K. Zhang et al.· Journal of the American Chem...· 0 citations
The rational design, synthesis, and mechanistic investigation of cyclopentene-based γ-aminobutyric acid analogues bearing alkyne or nitrile warheads as potent hOAT inactivators are reported, expanding the mechanistic repertoire of PLP-dependent enzyme inactivation and providing a generalizable framework for designing highly selective mechanism-based inactivators.
Feng Wang, M. Corrigan, N. Le et al.· Journal of the American Chem...· 0 citations
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