Human copper-dependent amine oxidases

Abstract

Copper amine oxidases (CAOs) are a class of enzymes that contain Cu(2+) and a tyrosine-derived quinone cofactor, catalyze the conversion of a primary amine functional group to an aldehyde, and generate hydrogen peroxide and ammonia as byproducts. These enzymes can be classified into two non-homologous families: 2,4,5-trihydroxyphenylalanine quinone (TPQ)-dependent CAOs and the lysine tyrosylquinone (LTQ)-dependent lysyl oxidase (LOX) family of proteins. In this review, we will focus on recent developments in the field of research concerning human CAOs and the LOX family of proteins. The aberrant expression of these enzymes is linked to inflammation, fibrosis, tumor metastasis/invasion and other diseases. Consequently, there is a critical need to understand the functions of these proteins at the molecular level, so that strategies targeting these enzymes can be developed to combat human diseases.

Keywords: Copper amine oxidase; Lysyl oxidase; Quinoprotein.

Figure 1. Structures of TPQ and LTQ

Tyrosine-derived quinone cofactors of human CAOs. TPQ (left) is derived from a conserved Tyr residue in CAOs, while LTQ (right) is derived from conserved Tyr and Lys residues in LOX. From (13,14).

Figure 2. DPQ as a common intermediate in the biogenesis of TPQ and LTQ

The proposed mechanisms for the biogenesis of the TPQ and LTQ cofactors share a dopaquinone (DPQ) intermediate. The 1,4-addition of either water (Pathway 1) or the ε-amino side chain of a peptidyl lysine residue (Pathway 2) to DPQ yields TPQ or LTQ, respectively. RCH₂NH₂ represents the side chain of a peptidyl lysine residue (-CH₂CH₂CH₂CH₂NH₂) within the polypeptide of a LOX family member. Adapted from (13,14).

Figure 3. Three different conformations of the TPQ ring detected in crystals of Arthrobacter globiformis amine oxidase (AGAO)

(a) In the active “off-copper” conformation, O4 of TPQ_ox is hydrogen-bonded to Tyr284. The reactive carbonyl group at C5 faces the substrate entry channel (Tyr296 in purple is located at the base of the proposed substrate channel) and the active site base (Asp298)(PDB accession number: 1IU7). (b) In the inactive “flipped” conformation of TPQ_ox, the TPQ ring has flipped 180° from the active conformation at the C2 axis. Consequently, the reactive carbonyl at C5 faces away from the substrate entry channel and Asp298 (PDB: 1AV4). (c) In the inactive “on-copper” conformation, O4 of TPQ_ox is directly ligated to Cu²⁺, where the C5 carbonyl group resides away from Asp298 and the substrate channel (PDB: 1AVL). Cu²⁺ is represented by an orange sphere, water molecules are represented by small blue spheres, the three His residues constituting the copper-binding site are in gray, hydrogen bonding interactions are represented by blue lines, and ligand interactions are represented by purple lines. Adapted from (7).

Figure 4. Active-site structures of mature D298K-AGAO and the putative DPQ intermediate detected during snapshot analysis of TPQ biogenesis in WT-AGAO

(A) DPQ intermediate (PDB: 1IVV), (B) D298K (PDB: 2YX9). Cu²⁺ is shown as an orange sphere, water molecules are shown as light-blue spheres, hydrogen bonding interactions are represented by blue lines, and ligand interactions are represented by purple lines. Val282 and Asn381 (white) form the edges of a wedge-shaped pocket. Hydrogen bonding distances are denoted in angstroms. From (22).

Figure 5. A proposed mechanism for the oxidation of an amine substrate by CAO

Scheme showing the classical ping-pong mechanism by which a copper amine oxidase oxidizes primary amines. Covalent intermediates are formed between TPQ and amines, in addition to oxidoreduction reactions of the TPQ cofactor. Adapted from (7).

Figure 6. Detailed proposed mechanism of the reductive half-reaction

The protonation states of the substrate and reaction intermediates are carefully controlled to achieve the optimal activity. The conserved Asp in the active site serves as a proton sink. The protonated substrate amine (RCH₂NH₃⁺) binds to the active site and is deprotonated by the Asp residue (which is deprotonated in the resting state). The reaction proceeds through two Schiff bases, i.e. a substrate Schiff base (TPQ_ssb, E) and a product Schiff base (TPQ_psb, G). TPQ_psb is mono-protonated to undergo facile hydrolysis to yield TPQ-aminoresourcinol (TPQ_red, J and K). In some amine oxidases, disproportionation reactions between TPQ_red and Cu²⁺ yield a semiquinone form of TPQ (TPQ_sq, L). From (8,24).

Figure 7

Phenylhydrazine (PH) adducts of TPQ and LTQ

Figure 8. LOX family of proteins

Cartoon summarizing the features of the LOX family of proteins. Each member retains a conserved C-terminal lysyl oxidase-like catalytic domain, which encompasses a copper-binding site composed of a His-X-His-X-His sequence, as well as a covalently bound LTQ cofactor formed from the linkage of conserved Tyr and Lys residues in the catalytic core. Each member also possesses an N-terminal secretion signal, and LOX and LOXL2-4 also contain predicted N-linked glycosylation sites (i.e. Asn-X-Ser/Thr). LOX and LOXL1 constitute one subfamily, possessing an N-terminal propeptide sequence, which is proteolytically removed by procollagen c-proteinase (BMP-1) at a conserved site (black arrow). LOXL2-4 constitute the other subfamily, with four SRCR domains instead of a propeptide.