Identification of an evolutionary conserved structural loop that is required for the enzymatic and biological function of tryptophan 2,3-dioxygenase

Abstract

The enzyme TDO (tryptophan 2,3-dioxygenase; TDO-2 in Caenorhabditis elegans) is a potential therapeutic target to cancer but is also thought to regulate proteotoxic events seen in the progression of neurodegenerative diseases. To better understand its function and develop specific compounds that target TDO we need to understand the structure of this molecule. In C. elegans we compared multiple different CRISPR/Cas9-induced tdo-2 deletion mutants and identified a motif of three amino acids (PLD) that is required for the enzymatic conversion of tryptophan to N-formylkynurenine. Loss of TDO-2’s enzymatic activity in PDL deletion mutants was accompanied by an increase in motility during aging and a prolonged lifespan, which is in line with the previously observed phenotypes induced by a knockdown of the full enzyme. Comparison of sequence structures suggests that blocking this motif might interfere with haem binding, which is essential for the enzyme’s activity. The fact that these three residues are situated in an evolutionary conserved structural loop of the enzyme suggests that the findings can be translated to humans. The identification of this specific loop region in TDO-2–essential for its catalytic function–will aid in the design of novel inhibitors to treat diseases in which the TDO enzyme is overexpressed or hyperactive.

Proteomic Characterization of Caenorhabditis elegans Larval Development

“…For example, the tryptophan‐converting enzyme tryptophan 2,3‐dioxygenase (TDO‐2) was identified in cluster C18, showing an increasing abundance during development. In support of our data showing an increased abundance of TDO‐2 during larval development, TDO‐2 has been shown previously to regulate adult life span and motility during ageing, and it has an age‐accumulative effect in regulating motility . In addition, we found that protein groups that tended to decrease in abundance during development were mainly associated with the actin cytoskeleton and cell cycle regulation.…”

Role of IDO and TDO in Cancers and Related Diseases and the Therapeutic Implications

“…Its heme group is present as an inactive Fe 3+ form that needs to be changed to Fe 2+ form to be active24. Furthermore, TDO is mainly found as homodimers that can transform to active tetramers of ∼190 kDa in eukaryotes and ∼120 kDa in prokaryotes 27, 33. Lewis-Ballester et al 34 found a binding site for L-Trp in hTDO that did not influence enzyme catalysis but can block the degradation of hTDO, revealing a new L-Trp-mediated regulation mechanism for cellular hTDO degradation.…”

“…Lewis-Ballester et al 34 found a binding site for L-Trp in hTDO that did not influence enzyme catalysis but can block the degradation of hTDO, revealing a new L-Trp-mediated regulation mechanism for cellular hTDO degradation. Additionally, Michels et al 33 found that three amino acid residues, TDO-2 (ΔPLD), TDO-2 (del), and TDO-2 (del B), were vital for the enzymatic activity of TDO.…”

Ommochromes in invertebrates: biochemistry and cell biology

“…TDO is a tetrameric complex containing a heme that is important for tryptophan oxidation (Capece et al, 2010). Three highly conserved loops are required for binding the heminic cofactor and for the induced-fit mechanism involved in binding tryptophan (Huang et al, 2013;Michels et al, 2016). Information on conserved sequences, structures and mechanisms are of great importance in the synthesis of new inhibitors of this enzymatic step (Huang et al, 2013;Michels et al, 2016).…”

“…Three highly conserved loops are required for binding the heminic cofactor and for the induced‐fit mechanism involved in binding tryptophan (Huang et al, ; Michels et al, ). Information on conserved sequences, structures and mechanisms are of great importance in the synthesis of new inhibitors of this enzymatic step (Huang et al, ; Michels et al, ). Hence, crystallographic data are needed if one intends to inhibit enzymes in both non‐model organisms and model organisms for which no tryptophan pathway mutants are available.…”