Lysine Tyrosylquinone Research Articles

Conformation Changes Associated with the Formation and Activation of Lysine Tyrosylquinone of LOXL2 Studied by Metadynamics Simulation

AbstractLysyl oxidase‐like 2 (LOXL2) is a Cu2+‐dependent amine oxidase that catalyzes the oxidative deamination of lysine residues of elastin and collagen to generate aldehyde groups. In the active state of LOXL2, its conserved catalytic domain has a lysine tyrosylquinone (LTQ) cofactor and a copper ion. LTQ is post‐translationally derived from Lys653 and Tyr689 of LOXL2. In this study, well‐tempered metadynamics simulations are used to investigate the conformation modulation from the inactive state to active state and obtain the free energy landscape. Interestingly, the simulations show that LTQ precursor residues cannot get close in Zn2+−bound LOXL2, whereas the copper ion binding triggers a conformation change to form an optional structure for LTQ biosynthesis. It is found that a prominent loop, consisting of residues 654–658, hinders the formation of LTQ in Zn2+−LOXL2 by preventing Lys653 from moving into active site of LOXL2, whereas this loop is relatively flexible in Cu2+−LOXL2. After the formation of LTQ, it needs to be activated by adjusting its position to be exposed to the solvent, thus allowing it to react with its substrate proteins. The results indicate that this process involves an intermediate state and that the activation of LTQ overcomes an energy barrier of 5.9 kcal mol−1.

LOXL2 in Cancer: A Two-Decade Perspective.

Lysyl Oxidase Like 2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises five lysine tyrosylquinone (LTQ)-dependent copper amine oxidases in humans. In 2003, LOXL2 was first identified as a promoter of tumour progression and, over the course of two decades, numerous studies have firmly established its involvement in multiple cancers. Extensive research with large cohorts of human tumour samples has demonstrated that dysregulated LOXL2 expression is strongly associated with poor prognosis in patients. Moreover, investigations have revealed the association of LOXL2 with various targets affecting diverse aspects of tumour progression. Additionally, the discovery of a complex network of signalling factors acting at the transcriptional, post-transcriptional, and post-translational levels has provided insights into the mechanisms underlying the aberrant expression of LOXL2 in tumours. Furthermore, the development of genetically modified mouse models with silenced or overexpressed LOXL2 has enabled in-depth exploration of its in vivo role in various cancer models. Given the significant role of LOXL2 in numerous cancers, extensive efforts are underway to identify specific inhibitors that could potentially improve patient prognosis. In this review, we aim to provide a comprehensive overview of two decades of research on the role of LOXL2 in cancer.

Insight into the Spatial Arrangement of the Lysine Tyrosylquinone and Cu2+ in the Active Site of Lysyl Oxidase-like 2.

Lysyl oxidase-2 (LOXL2) is a Cu2+ and lysine tyrosylquinone (LTQ)-dependent amine oxidase that catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine residues to promote crosslinking of extracellular matrix proteins. LTQ is post-translationally derived from Lys653 and Tyr689, but its biogenesis mechanism remains still elusive. A 2.4 Å Zn2+-bound precursor structure lacking LTQ (PDB:5ZE3) has become available, where Lys653 and Tyr689 are 16.6 Å apart, thus a substantial conformational rearrangement is expected to take place for LTQ biogenesis. However, we have recently shown that the overall structures of the precursor (no LTQ) and the mature (LTQ-containing) LOXL2s are very similar and disulfide bonds are conserved. In this study, we aim to gain insights into the spatial arrangement of LTQ and the active site Cu2+ in the mature LOXL2 using a recombinant LOXL2 that is inhibited by 2-hydrazinopyridine (2HP). Comparative UV-vis and resonance Raman spectroscopic studies of the 2HP-inhibited LOXL2 and the corresponding model compounds and an EPR study of the latter support that 2HP-modified LTQ serves as a tridentate ligand to the active site Cu2. We propose that LTQ resides within 2.9 Å of the active site of Cu2+ in the mature LOXL2, and both LTQ and Cu2+ are solvent-exposed.

A 3D-Predicted Structure of the Amine Oxidase Domain of Lysyl Oxidase-Like 2.

Lysyl oxidase-like 2 (LOXL2) has been recognized as an attractive drug target for anti-fibrotic and anti-tumor therapies. However, the structure-based drug design of LOXL2 has been very challenging due to the lack of structural information of the catalytically-competent LOXL2. In this study; we generated a 3D-predicted structure of the C-terminal amine oxidase domain of LOXL2 containing the lysine tyrosylquinone (LTQ) cofactor from the 2.4Å crystal structure of the Zn2+-bound precursor (lacking LTQ; PDB:5ZE3); this was achieved by molecular modeling and molecular dynamics simulation based on our solution studies of a mature LOXL2 that is inhibited by 2-hydrazinopyridine. The overall structures of the 3D-modeled mature LOXL2 and the Zn2+-bound precursor are very similar (RMSD = 1.070Å), and disulfide bonds are conserved. The major difference of the mature and the precursor LOXL2 is the secondary structure of the pentapeptide (His652-Lys653-Ala654-Ser655-Phe656) containing Lys653 (the precursor residue of the LTQ cofactor). We anticipate that this peptide is flexible in solution to accommodate the conformation that enables the LTQ cofactor formation as opposed to the β-sheet observed in 5ZE3. We discuss the active site environment surrounding LTQ and Cu2+ of the 3D-predicted structure.

Mass Spectrometry-Based Disulfide Mapping of Lysyl Oxidase-like 2.

Lysyl oxidase-like 2 (LOXL2) catalyzes the oxidative deamination of peptidyl lysines and hydroxylysines to promote extracellular matrix remodeling. Aberrant activity of LOXL2 has been associated with organ fibrosis and tumor metastasis. The lysine tyrosylquinone (LTQ) cofactor is derived from Lys653 and Tyr689 in the amine oxidase domain via post-translational modification. Based on the similarity in hydrodynamic radius and radius of gyration, we recently proposed that the overall structures of the mature LOXL2 (containing LTQ) and the precursor LOXL2 (no LTQ) are very similar. In this study, we conducted a mass spectrometry-based disulfide mapping analysis of recombinant LOXL2 in three forms: a full-length LOXL2 (fl-LOXL2) containing a nearly stoichiometric amount of LTQ, Δ1-2SRCR-LOXL2 (SRCR1 and SRCR2 are truncated) in the precursor form, and Δ1-3SRCR-LOXL2 (SRCR1, SRCR2, SRCR3 are truncated) in a mixture of the precursor and the mature forms. We detected a set of five disulfide bonds that is conserved in both the precursor and the mature recombinant LOXL2s. In addition, we detected a set of four alternative disulfide bonds in low abundance that is not associated with the mature LOXL2. These results suggest that the major set of five disulfide bonds is retained post-LTQ formation.

2‐Hydrazinopyridine Inhibition of Lysyl oxidase‐like 2

Lysyl oxidase-like 2 (LOXL2) is associated with tumor metastasis/invasion and organ fibrosis via promoting the extracellular matrix (ECM) stiffening. LOXL2 is a Cu2+ and lysine tyrosylquinone (LTQ) dependent amine oxidase and known to catalyze the oxidative deamination of peptidyl lysine and hydroxyl lysine residues in ECM proteins (e.g. collagens and elastin). The LTQ cofactor of LOXL2 is posttranslationaly derived from peptidyl Lys and Tyr residues. Recently, an X-ray structure of a Zn2+-bound precursor of LOXL2 became available. Surprisingly, the precursor residues of the LTQ cofactor, Lys653 and Tyr689 were 16.6 Å apart, indicating that substantial conformational change is required for the biogenesis of the LTQ cofactor. A 3D homology modeling study of the catalytic domain of lysyl oxidase (LOX) based on the precursor structure of LOXL2 even suggested a possibility of disulfide bond shuffling during biogenesis. In this study, we focus on a catalytically-competent LOXL2 and aim to gain insight into the active site environment. Comparative UV-vis spectroscopic studies of LOXL2 inhibited with 2-hydrazinopyridine (2-HP) and model compound mimicking LTQ-2HP were conducted. We obtained a strong evidence to support that the LTQ cofactor resides within 2.8 Å from the active site Cu2+ and a large conformational change is not required for the formation of the LTQ cofactor.

Reaction mechanism of lysyl oxidase-like 2 (LOXL2) studied by computational methods

Lysyl oxidase-like 2 (LOXL2) is a copper-dependent amine oxidase that catalyzes the oxidative deamination of the ε-amino group of lysines/hydroxylysines on substrate proteins (collagen and elastin) to form aldehyde groups. The generated aldehyde groups are of significance in crosslinking with the adjacent aldehyde or ε-amino group on proteins in extracellular matrix. In this paper, we have studied the reaction mechanism of LOXL2 by means of quantum mechanics (QM) and combined QM and molecular mechanics (QM/MM) methods. This study is divided into two parts, i.e. the biosynthesis of lysine tyrosylquinone (LTQ) cofactor and oxidative deamination of ε-amino group of lysine by LTQ. For the former part, the reaction is driven by a large exothermicity of about 284 kJ/mol. Dopaquinone radical (DPQr) is suggested to be an intermediate state in this reaction. In addition, His652 residue is predicted to serve as proton acceptor. The rate-determining step for the biosynthesis of LTQ is found to be hydrogen-atom abstraction from the benzene ring on substrate by Cu2+-hydroxide, which is a proton-coupled electron transfer (PCET) process with an energy barrier of 84 kJ/mol. For the latter part, the reaction is exothermic by about 145 kJ/mol, and the copper ion is proposed to play a role of redox catalyst in the last step to generate the product of aldehyde. However, the copper ion might not be indispensable for the latter part, which is consistent with the previous study.

Lysyl oxidases: from enzyme activity to extracellular matrix cross-links.

The lysyl oxidase family comprises five members in mammals, lysyl oxidase (LOX) and four lysyl oxidase like proteins (LOXL1-4). They are copper amine oxidases with a highly conserved catalytic domain, a lysine tyrosylquinone cofactor, and a conserved copper-binding site. They catalyze the first step of the covalent cross-linking of the extracellular matrix (ECM) proteins collagens and elastin, which contribute to ECM stiffness and mechanical properties. The role of LOX and LOXL2 in fibrosis, tumorigenesis, and metastasis, including changes in their expression level and their regulation of cell signaling pathways, have been extensively reviewed, and both enzymes have been identified as therapeutic targets. We review here the molecular features and three-dimensional structure/models of LOX and LOXLs, their role in ECM cross-linking, and the regulation of their cross-linking activity by ECM proteins, proteoglycans, and by inhibitors. We also make an overview of the major ECM cross-links, because they are the ultimate molecular readouts of LOX/LOXL activity in tissues. The recent 3D model of LOX, which recapitulates its known structural and biochemical features, will be useful to decipher the molecular mechanisms of LOX interaction with its various substrates, and to design substrate-specific inhibitors, which are potential antifibrotic and antitumor drugs.

A Three-Dimensional Model of Human Lysyl Oxidase, a Cross-Linking Enzyme.

Lysyl oxidase (LOX) is a cross-linking enzyme identified 50 years ago, but its 3D structure is still unknown. We have thus built a 3D model of human LOX by homology modeling using the X-ray structure of human lysyl oxidase-like 2 as a template. This model is the first one to recapitulate all known biochemical features of LOX, namely, the coordination of the copper ion and the formation of the lysine tyrosylquinone cofactor and the disulfide bridges. Furthermore, this model is stable during a 1 μs molecular dynamics simulation. The catalytic site is located in a groove surrounded by two loops. The distance between these loops fluctuated during the simulations, which suggests that the groove forms a hinge with a variable opening, which is able to accommodate the various sizes of LOX substrates. This 3D model is a pre-requisite to perform docking experiments with LOX substrates and other partners to identify binding sites and to design new LOX inhibitors specific for therapeutic purpose.

Protein-Derived Cofactors Revisited: Empowering Amino Acid Residues with New Functions.

A protein-derived cofactor is a catalytic or redox-active site in a protein that is formed by post-translational modification of one or more amino acid residues. These post-translational modifications are irreversible and endow the modified amino acid residues with new functional properties. This Perspective focuses on the following advances in this area that have occurred during recent years. The biosynthesis of the tryptophan tryptophylquinone cofactor is catalyzed by a diheme enzyme, MauG. A bis-FeIV redox state of the hemes performs three two-electron oxidations of specific Trp residues via long-range electron transfer. In contrast, a flavoenzyme catalyzes the biosynthesis of the cysteine tryptophylquinone (CTQ) cofactor present in a newly discovered family of CTQ-dependent oxidases. Another carbonyl cofactor, the pyruvoyl cofactor found in classes of decarboxylases and reductases, is formed during an apparently autocatalytic cleavage of a precursor protein at the N-terminus of the cleavage product. It has been shown that in at least some cases, the cleavage is facilitated by binding to an accessory protein. Tyrosylquinonine cofactors, topaquinone and lysine tyrosylquinone, are found in copper-containing amine oxidases and lysyl oxidases, respectively. The physiological roles of different families of these enzymes in humans have been more clearly defined and shown to have significant implications with respect to human health. There has also been continued characterization of the roles of covalently cross-linked amino acid side chains that influence the reactivity of redox-active metal centers in proteins. These include Cys-Tyr species in galactose oxidase and cysteine dioxygenase and the Met-Tyr-Trp species in the catalase-peroxidase KatG.

Human lysyl oxidase-like 2

Lysyl oxidase like-2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises Cu2+- and lysine tyrosylquinone (LTQ)-dependent amine oxidases. LOXL2 is proposed to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 has also been proposed to regulate extracellular and intracellular cell signaling pathways. Dysregulation of LOXL2 has been linked to many diseases, including cancer, pro-oncogenic angiogenesis, fibrosis and heart diseases. In this review, we will give an overview of the current understandings and hypotheses regarding the molecular functions of LOXL2.

Human copper-dependent amine oxidases

Copper amine oxidases (CAOs) are a class of enzymes that contain Cu2+ 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.

Investigation into Structural Changes of the Copper Binding Site in Lysyl Oxidase upon Substrate and Inhibitor Docking

The present paper reports a computational investigation of potential communication between the lysine tyrosylquinone (LTQ) and copper cofactors within lysyl oxidase (LOX). Various substrates and inhibitors of LOX were docked into the active site in our computer generated model of the enzyme. Conformational changes in the vicinity of the copper site as well as changes in the electrostatic environment were identified. The appearance of a canal-like structure involving tyrosine 35 (TYR35) and glutamine 104 (GLN104) residues was shown to be consistent upon docking of a variety of different compounds. Interactions between LOX and its natural substrate, collagen, were also explored through molecular dynamic simulations. The possibility of communication between the organic and inorganic cofactors in LOX was proposed, aiding the ongoing debate regarding the role of copper in the catalytic mechanism of this important enzyme.

Post-translational Modifications of Recombinant Human Lysyl Oxidase-like 2 (rhLOXL2) Secreted from Drosophila S2 Cells*

Human lysyl oxidase-like 2 (hLOXL2) is highly up-regulated in metastatic breast cancer cells and tissues and induces epithelial-to-mesenchymal transition, the first step of metastasis/invasion. hloxl2 encodes four N-terminal scavenger receptor cysteine-rich domains and the highly conserved C-terminal lysyl oxidase (LOX) catalytic domain. Here, we assessed the extent of the post-translational modifications of hLOXL2 using truncated recombinant proteins produced in Drosophila S2 cells. The recombinant proteins are soluble, in contrast to LOX, which is consistently reported to require 2-6 m urea for solubilization. The recombinant proteins also show activity in tropoelastin oxidation. After phenylhydrazine derivatization and trypsin digestion, we used mass spectrometry to identify peptides containing the derivatized lysine tyrosylquinone cross-link at Lys-653 and Tyr-689, as well as N-linked glycans at Asn-455 and Asn-644. Disruption of N-glycosylation by site-directed mutagenesis or tunicamycin treatment completely inhibited secretion so that only small quantities of inclusion bodies were detected. The N-glycosylation site at Asn-644 in the LOX catalytic domain is not conserved in human LOX (hLOX), although the LOX catalytic domain of hLOX shares ∼50% identity and ∼70% homology with hLOXL2. The catalytic domain of hLOX was not secreted from S2 cells using the same expression system. These results suggest that the N-glycan at Asn-644 of hLOXL2 enhances the solubility and stability of the LOX catalytic domain.

Intracellular and extracellular human lysyl oxidase‐like 2 in metastatic breast cancer cells

Human lysyl oxidase‐like 2 (HLOXL2) is highly up‐regulated in metastatic breast cancer cells and tissues. Native and recombinant HLOXL2s (rHLOXL2s) are soluble and mostly secreted into the media, but also found in cytosol and nucleus. Secreted HLOXL2 (~100 kDa) was confirmed for the presence of glycosylation, proteolysis, and formation of lysine tyrosylquinone cofactor. The fourth SRCR and catalytic domains of HLOXL2 are in communication to achieve optimal activity, and N‐glycosylation at Asn455 and Asn644 is essential for protein secretion. MCF‐7 cells stably expressing rHLOXL2s show higher proliferation and invasiveness in vitro, but do not exhibit characteristic metastasis molecular markers. Addition of β‐Aminopropionitrile, the specific inhibitor of HLOXL2, significantly abolished proliferation and invasiveness, as in the case for MDA‐MB‐231. Intracellular HLOXL2 (~80 kDa) is unglycosylated, but catalytically competent. Wild type MCF‐7 cells can uptake the purified secreted rHLOXL2s, and the exogenous N‐glycosylated rHLOXL2s localize in the nucleus and induce epithelial‐to‐mesenchymal transition. Evidence supports that HLOXL2 goes beyond its traditional role of initiating collagen and elastin cross‐linking extracellularly, its intracellular accumulation leads to a dramatic increase of metastasis/invasion in breast cancer cells.

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

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.

Quinoproteins and Cofactors: Expecting the Unexpected

Naturally occurring modifications of the standard 20 amino acids expand the repertoire of functional groups available for catalysis within enzyme active sites. A novel class of enzymatic quino-cofactors has been identified over the past decade. These are derived from protein and peptide-bound side chains and include the eukaryotic topa quinone (TPQ) and lysine tyrosyl quinone (LTQ) cofactors, together with the prokaryotic tryptophan tryptophyl quinone (TTQ), cysteine tryptophyl quinone (CTQ) and pyrroloquinoline quinine (PQQ). As an exception to the protein-bound quino-cofactors (TPQ, LTQ, TTQ and CTQ), the precursor to PQQ is generated within a polypeptide chain, followed by its release via proteolysis to yield a freely dissociable cofactor. This presentation will trace the history of the field, followed by a comparison of the properties for TPQ biogenesis (and catalysis) to PQQ biogenesis - with the former occurring via a self-processing mechanism while the latter requires multiple gene products. Many unexpected features have emerged from these studies that include the occurrence of multiple catalytic functions within single enzyme active sites, a non-redox role for a redox active metal center, and the control of molecular oxygen reactivity via non-metal, protein-derived binding pockets. (Supported by NIH GM25765 and GM39296)

Involvement of a Putative [Fe-S]-cluster-binding Protein in the Biogenesis of Quinohemoprotein Amine Dehydrogenase

Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallestgamma-subunit of the alphabetagamma heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the alpha- and gamma-subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The alpha- and beta-subunits were both detected in the periplasm of the mutant strain, whereas the gamma-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the gamma-subunit isolated from the mutant strain had not undergone posttranslational modification. These results unequivocally show that the putative [Fe-S]-cluster- and SAM-binding ORF2 protein is necessary for the posttranslational processing of gamma-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links.

The Formation of Lysine Tyrosylquinone (LTQ) Is a Self-Processing Reaction. Expression and Characterization of a Drosophila Lysyl Oxidase

Recent work in our laboratory has established methods for the expression and purification of a recombinant form of Drosophila lysyl oxdidase (rDMLOXL-1) [Molnar, J., Ujfaludi, Z., Fong, S. F. T., Bollinger, J. A., Waro, G., Fogelgren, B., Dooley, D. M., Mink, M., and Csiszar, K. (2005) J. Biol. Chem. 280, 22977-22985]. Previous investigations on the expression and purification of recombinant forms of lysyl oxidase [Kagan, H. M., Reddy, V. B., Panchenko, M. V., Nagan, N., Boak, A. M., Gacheru, S. N., and Thomas, K. (1995) J. Cell. Biochem. 59, 329-338] and lysyl oxidase-like proteins [Jung, S. T., Kim, M. S., Seo, J. Y., Kim, H. C., and Kim, Y. (2003) Protein Expression Purif. 31, 240-246] [Molnar, J., Fong, K. S. K., He, Q. P., Hayashi, K., Kim, Y., Fong, S. F. T., Fogelgren, B., Szauter, K. M., Mink, M., and Csiszar, K. (2003) Biochim. Biophys. Acta 1647, 220-224] have been reported in the literature. However, this is the first time that an expression system has been developed yielding sufficient amounts of a recombinant lysyl oxidase for detailed characterization. rDmLOXL-1 is secreted into the medium from S2 cells, and the protein is readily purified by Cibacon blue affinity chromatography yielding 10 mg of protein per liter of medium. The protein, as initially purified, is inactive and has no detectable copper or cofactor present. Following aerobic dialysis against copper, the protein is active and displays an electronic absorption spectrum with lambda(max) at 504 nm, consistent with the presence of an organic cofactor. Addition of phenylhydrazine to the copper-loaded protein produced a high-affinity adduct with lambda(max) at 454 nm. Comparison of the resonance Raman spectra of this adduct and a phenylhydrazine-labeled model compound of lysine tyrosylquinone (LTQ) establishes that the cofactor in the active, copper-containing enzyme is LTQ. Collectively, the data demonstrate that LTQ biogenesis most likely occurs by self-processing chemistry, requiring only the precursor protein, copper, and oxygen. Electron paramagnetic resonance and circular dichroism spectroscopy were used to characterize the Cu(II) site in rDmLOXL-1. The data are consistent with a tetragonal Cu(II) site with nitrogen and oxygen ligands. Recombinant DmLOXL-1 displayed significant activity toward tropoelastin and a wide variety of amines including polyamines and diamines. beta-aminoproprionitrile (betaAPN), a well-known irreversible inhibitor of mammalian lysyl oxidases, is also a potent inhibitor of rDmLOXL-1. Results from this investigation have important implications for the lysyl oxidase family.

Flavoenzyme catalysed oxidation of amines: roles for flavin and protein-based radicals

Amines are a carbon source for the growth of a number of bacterial species and they also play key roles in neurotransmission, cell growth and differentiation, and neoplastic cell proliferation. Enzymes have evolved to catalyse these reactions and these oxidoreductases can be grouped into the flavoprotein and quinoprotein families. The mechanism of amine oxidation catalysed by the quinoprotein amine oxidases is understood reasonably well and occurs through the formation of enzyme-substrate covalent adducts with TPQ (topaquinone), TTQ (tryptophan tryptophylquinone), CTQ (cysteine tryptophylquinone) and LTQ (lysine tyrosyl quinone) redox centres. Oxidation of amines by flavoenzymes is less well understood. The role of protein-based radicals and flavin semiquinone radicals in the oxidation of amines is discussed.