Over-expression of lysyl oxidase-like 2 (LOXL2) is associated with several hepatic and vascular fibrotic diseases and tumor progression in some aggressive cancers. Secreted LOXL2 promotes extracellular matrix crosslinking by catalyzing the oxidative deamination of peptidyl lysine. A great deal remains to be learned about the post-translational modifications of LOXL2, including whether such modifications modulate enzymatic and disease-promoting activities; such knowledge would inform the development of potential therapies. We discovered that upon secretion in cell culture, LOXL2 undergoes proteolytic processing of the first two of four scavenger receptor cysteine-rich domains at the N-terminus. A similar pattern of processing was also evident in tissue extracts from an invasive ductal carcinoma patient. Processing occurred at 314Arg-315Phe-316Arg-317Lys↓−318Ala-, implicating proprotein convertases. siRNA-mediated knockdown of proprotein convertases (furin, PACE4, and PC5/6), as well as incubation with their recombinant forms, showed that PACE4 is the major protease that acts on extracellular LOXL2. Unlike LOX, which requires cleavage of its propeptide for catalytic activation, cleavage of LOXL2 was not essential for tropoelastin oxidation nor for crosslinking of collagen type IV in vitro. However, in the latter case, processing enhanced the extent of collagen crosslinking ~2-fold at [LOXL2] ≤ 10 nM. These results demonstrate an important difference in the regulatory mechanisms for LOX and LOXL2 catalytic activity. Moreover, they pave the way for further studies of potential differential functions of LOXL2 isoforms in fibrosis and tumor progression.

A new class of poly-N-vinylpyrrolidinones containing an asymmetric center at C5 of the pyrrolidinone ring were synthesized from l-amino acids. The polymers, particularly 17, were used to stabilize nanoclusters such as Pd/Au for the catalytic asymmetric oxidations of 1,3- and 1,2-cycloalkanediols and alkenes, and Cu/Au was used for C-H oxidation of cycloalkanes. It was found that the bulkier the C5 substituent in the pyrrolidinone ring, the greater the optical yields produced. Both oxidative kinetic resolution of (±)-1,3- and 1,2-trans-cycloalkanediols and desymmetrization of meso cis-diols took place with 0.15 mol % Pd/Au (3:1)-17 under oxygen atmosphere in water to give excellent chemical and optical yields of (S)-hydroxy ketones. Various alkenes were oxidized with 0.5 mol % Pd/Au (3:1)-17 under 30 psi of oxygen in water to give the dihydroxylated products in >93% ee. Oxidation of (R)-limonene at 25 °C occurred at the C-1,2-cyclic alkene function yielding (1S,2R,4R)-dihydroxylimonene 49 in 92% yield. Importantly, cycloalkanes were oxidized with 1 mol % Cu/Au (3:1)-17 and 30% HO in acetonitrile to afford chiral ketones in very good to excellent chemical and optical yields. Alkene function was not oxidized under the reaction conditions. Mechanisms were proposed for the oxidation reactions, and observed stereo- and regio-chemistry were summarized.

Protonation reactions involving organometallic complexes are ubiquitous in redox chemistry and often result in the generation of reactive metal hydrides. However, some organometallic species supported by η 5 -pentamethylcyclopentadienyl (Cp*) ligands have recently been shown to undergo ligand-centered protonation by direct proton transfer from acids or tautomerization of metal hydrides, resulting in the generation of complexes bearing the uncommon η 4 -pentamethylcyclopentadiene (Cp*H) ligand. Here, time-resolved pulse radiolysis (PR) and stopped-flow spectroscopic studies have been applied to examine the kinetics and atomistic details involved in the elementary electron- and proton-transfer steps leading to complexes ligated by Cp*H, using Cp*Rh(bpy) as a molecular model (where bpy is 2,2′-bipyridyl). Stopped-flow measurements coupled with infrared and UV-visible detection reveal that the sole product of initial protonation of Cp*Rh(bpy) is [Cp*Rh(H)(bpy)] + , an elusive hydride complex that has been spectroscopically and kinetically characterized here. Tautomerization of the hydride leads to the clean formation of [(Cp*H)Rh(bpy)] + . Variable-temperature and isotopic labeling experiments further confirm this assignment, providing experimental activation parameters and mechanistic insight into metal-mediated hydride-to-proton tautomerism. Spectroscopic monitoring of the second proton transfer event reveals that both the hydride and related Cp*H complex can be involved in further reactivity, showing that [(Cp*H)Rh] is not necessarily an off-cycle intermediate, but, instead, depending on the strength of the acid used to drive catalysis, an active participant in hydrogen evolution. Identification of the mechanistic roles of the protonated intermediates in the catalysis studied here could inform design of optimized catalytic systems supported by noninnocent cyclopentadienyl-type ligands.

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.

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.