Author:
Kim Sung-Mi,Liu Yi,Wang YongQiang,Karkashon Shay,Lewis-Ballester Ariel,Yeh Syun-Ru,Correia Maria Almira
Abstract
AbstractHepatic tryptophan 2,3-dioxygenase (TDO) is a cytoplasmic homotetrameric hemoprotein and the rate-limiting enzyme in the irreversible degradation of the essential amino acidL-tryptophan (L-Trp) to N-formylkynurenine, thus controlling the flux ofL-Trp into its serotonergic and kynureninic/NAD pathways. TDO has long been recognized to be substrate-inducible via protein stabilization, but the molecular mechanism of this stabilization has remained elusive. Recent elucidation of human TDO (hTDO) crystal structure has identified a high-affinity (Kd ≈ 0.5 μM) Trp-binding exosite in each of its 4 monomeric subunits. Mutation of the Glu105, Trp208and Arg211comprising this exosite not only abolished the high-affinityL-Trp binding, but also accelerated the ubiquitin-dependent proteasomal degradation of hTDO. We have further characterized this hTDO degradation by documenting that its ubiquitination by gp78/AMFR and CHIP E2/E3 ligase complexes occurs on external Lys-residues within or vicinal to acidic Asp/Glu and phosphorylated pSer/pThr (DEpSpT)-clusters. Furthermore, we have identified the unstructured hTDO N- and C-termini as imparting relatively high proteolytic instability, as their deletion (ΔNC) markedly prolonged hTDO t1/2. Additionally, although previous studies reported that upon hepatic heme-depletion, the heme-free apoTDO turns over with a t1/2≈ 2.2 h relative to the t1/2of 7.7 h of holoTDO, mutating the axial heme-ligating His328to Ala has the opposite effect of prolonging hTDO t1/2. Most importantly, introducing the exosite mutation into the ΔNC-deleted or H328A-mutant completely abolished their prolonged half-lives irrespective ofL-Trp presence or absence, thereby revealing that the exosite is the molecular lynchpin that definesL-Trp-mediated TDO induction via protein stabilization.
Publisher
Cold Spring Harbor Laboratory
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5. Heme-Containing Oxygenases
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