Trapping of a Dopaquinone Intermediate in the TPQ Cofactor Biogenesis in a Copper-Containing Amine Oxidase fromArthrobacterglobiformis

Abstract

The biogenesis of the topaquinone (TPQ) cofactor of copper amine oxidase (CAO) is self-catalyzed and requires copper and molecular oxygen. A dopaquinone intermediate has been proposed to undergo 1,4-addition of a copper-associated water molecule to form the reduced form of TPQ (TPQ(red)), followed by facile oxidation by O(2) to yield the mature TPQ (TPQ(ox)). In this study, we have incorporated a lysine residue in the active site of Arthrobacter globiformis CAO (AGAO) by site-directed mutagenesis to produce D298K-AGAO. The X-ray crystal structure of D298K-AGAO at 1.7-A resolution revealed that a covalent linkage formed between the epsilon-amino side chain of Lys298 and the C2 position of a dopaquinone derived from Tyr382, a precursor to TPQ(ox). We assigned the species as an iminoquinone tautomer (LTI) of lysine tyrosylquinone (LTQ), the organic cofactor of lysyl oxidase (LOX). The time course of the formation of LTI at pH 6.8 was followed by UV/vis and resonance Raman spectroscopies. In the early phase of the reaction, an LTQ-like intermediate was observed. This intermediate then slowly converted to LTI in an isosbestic manner. Not only is the presence of a dopaquinone intermediate in the TPQ biogenesis confirmed, but it also provides strong support for the proposed intermediacy of a dopaquinone in the biogenesis of LTQ in LOX. Further, this study indicates that the dopaquinone intermediate in AGAO is mobile and can swing from the copper site into the active-site wedge to react with Lys298.

Copper Active Sites in Biology

“…1195 In addition, crystal structures have been obtained of the HPAO variant Y305F, which reacts with O 2 but does not form the mature TPQ cofactor and instead shows a unique partially processed form of the substrate Tyr residue where the ring is multiply-peroxidated, 1190 and the AGAO variant D298K, which forms the LTQ cofactor that is present in lysyl oxidase. 1233 The overall structure of the AO enzyme in these forms is the same and the only significant differences are observed at the active site.…”

“…Another mutation performed in AGAO, D298K, allows trapping of covalet trapping of the dopaquinone intermediate in biogenesis. 1233 Nucleophilic attack of the variant Lys residue on the dopaquinone intermediate leads to formation of a crosslink similar to the one present in lysyl oxidase. This is proposed to be the mechanism for formation of LTQ, the lysyl oxidase cofactor, since lysyl oxidase does contain a Lys residue at the appropriate position in its sequence.…”

“…1233 Stabilization of the iminoquinol tautomer of LTQ (Figure 224, 10) over the aminoquinone form (Figure 224, 9) is influenced by hydrogen bonding from another second sphere residue, Y284, and so some of the second sphere residues in LOX must also play a role in the stabilization of the active form of the cofactor. These results establish a mechanistic proposal for LTQ biogenesis, where the early steps of the cofactor biogenesis reaction (Cu II substrate activation, O 2 attack to form a Cu II -peroxoquinone and O-O bond cleavage to form DPQ) are the same in both TPQ and LTQ biogenesis and the only difference is what nucleophile adds to the DPQ intermediate – Cu II -coordinated hydroxide (TPQ) or a Lys residue (LTQ biogenesis pathway, Figure 224).…”

Activation of dioxygen by copper metalloproteins and insights from model complexes

“…17) [87, 273–275]. The consensus mechanism starts with binding of O 2 to a pocket in the protein, which induces a conformational change leading to deprotonation and coordination of the “preprocessed” active site tyrosine (the preprocessed- or pro-enzyme refers to CAO before oxidation of tyrosine to TPQ) and formation of a Cu II tyrosinate complex (Fig.…”

MCF-7 Cells Expressing Nuclear Associated Lysyl Oxidase-like 2 (LOXL2) Exhibit an Epithelial-to-Mesenchymal Transition (EMT) Phenotype and Are Highly Invasive in Vitro

“…Presumably, Cu 2ϩ incorporation and LTQ biogenesis take place in the endoplasmic reticulum, which is a more oxidative environment than the cytosol (30). Model studies indicate that biogenesis occurs after the active site is prearranged (31,32).…”