The resurgence of interest in monoamine oxidases (MAOs) has been fueled by recent correlations of this enzymatic activity with cardiovascular, neurological, and oncological disorders. This has ...promoted increased research into selective MAO-A and MAO-B inhibitors. Here, we shed light on how selective inhibition of MAO-A and MAO-B can be achieved by geometric isomers of cis- and trans-1-propargyl-4-styrylpiperidines. While the cis isomers are potent human MAO-A inhibitors, the trans analogues selectively target only the MAO-B isoform. The inhibition was studied by kinetic analysis, UV–vis spectrum measurements, and X-ray crystallography. The selective inhibition of the MAO-A and MAO-B isoforms was confirmed ex vivo in mouse brain homogenates, and additional in vivo studies in mice show the therapeutic potential of 1-propargyl-4-styrylpiperidines for central nervous system disorders. This study represents a unique case of stereoselective activity of cis/trans isomers that can discriminate between structurally related enzyme isoforms.
UDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD+-dependent epimerase belonging to the short-chain ...dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase from Bacillus cereus. The geometry of the substrate-NAD+ interactions is finely arranged to promote hydride transfer. The exquisite complementarity between glucuronic acid and its binding site is highlighted by the observation that the unligated cavity is occupied by a cluster of ordered waters whose positions overlap the polar groups of the sugar substrate. Co-crystallization experiments led to a structure where substrate- and product-bound enzymes coexist within the same crystal. This equilibrium structure reveals the basis for a “swing and flip” rotation of the pro-chiral 4-keto-hexose-uronic acid intermediate that results from glucuronic acid oxidation, placing the C4′ atom in position for receiving a hydride ion on the opposite side of the sugar ring. The product-bound active site is almost identical to that of the substrate-bound structure and satisfies all hydrogen-bonding requirements of the ligand. The structure of the apoenzyme together with the kinetic isotope effect and mutagenesis experiments further outlines a few flexible loops that exist in discrete conformations, imparting structural malleability required for ligand rotation while avoiding leakage of the catalytic intermediate and/or side reactions. These data highlight the double nature of the enzymatic mechanism: the active site features a high degree of precision in substrate recognition combined with the flexibility required for intermediate rotation.
Following a hybridization strategy, a series of 5-substituted-1H-indazoles were designed and evaluated in vitro as inhibitors of human monoamine oxidase (hMAO) A and B. Among structural ...modifications, the bioisostere-based introduction of 1,2,4-oxadiazole ring returned the most potent and selective human MAO B inhibitor (compound 20, IC50 = 52 nM, SI > 192). The most promising inhibitors were studied in cell-based neuroprotection models of SH-SY5Y and astrocytes line against H2O2. Moreover, preliminary drug-like features (aqueous solubility at pH 7.4; hydrolytic stability at acidic and neutral pH) were assessed for selected 1,2,4-oxadiazoles and compared to amide analogues through RP-HPLC methods. Molecular docking simulations highlighted the crucial role of molecular flexibility in providing a better shape complementarity for compound 20 within MAO B enzymatic cleft than rigid analogue 18. Enzymatic kinetics analysis along with thermal stability curves (Tm shift = +2.9 °C) provided clues of a tight-binding mechanism for hMAO B inhibition by 20.
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•Twentyfive 1H-indazoles were designed and tested as hMAO inhibitors.•Bioisosteric amide replacement via 1,2,4-oxadiazoles was investigated.•Compound 20 was the most potent and selective MAO B inhibitor (IC50 = 52 nM, SI > 192).•20 protected both SH-SY5Y and astrocytes from H2O2 insult.•20 displayed tight-binder's features toward hMAO B (Tm shift = +2.9 °C).
F420H2-dependent enzymes reduce a wide range of substrates that are otherwise recalcitrant to enzyme-catalyzed reduction, and their potential for applications in biocatalysis has attracted increasing ...attention. Thermobifida fusca is a moderately thermophilic bacterium and holds high biocatalytic potential as a source for several highly thermostable enzymes. We report here on the isolation and characterization of a thermostable F420: NADPH oxidoreductase (Tfu-FNO) from T. fusca, the first F420-dependent enzyme described from this bacterium. Tfu-FNO was heterologously expressed in Escherichia coli, yielding up to 200 mg of recombinant enzyme per liter of culture. We found that Tfu-FNO is highly thermostable, reaching its highest activity at 65 °C and that Tfu-FNO is likely to act in vivo as an F420 reductase at the expense of NADPH, similar to its counterpart in Streptomyces griseus. We obtained the crystal structure of FNO in complex with NADP+ at 1.8 Å resolution, providing the first bacterial FNO structure. The overall architecture and NADP+-binding site of Tfu-FNO were highly similar to those of the Archaeoglobus fulgidus FNO (Af-FNO). The active site is located in a hydrophobic pocket between an N-terminal dinucleotide binding domain and a smaller C-terminal domain. Residues interacting with the 2′-phosphate of NADP+ were probed by targeted mutagenesis, indicating that Thr-28, Ser-50, Arg-51, and Arg-55 are important for discriminating between NADP+ and NAD+. Interestingly, a T28A mutant increased the kinetic efficiency >3-fold as compared with the wild-type enzyme when NADH is the substrate. The biochemical and structural data presented here provide crucial insights into the molecular recognition of the two cofactors, F420 and NAD(P)H by FNO.
Glycerol is a major byproduct of biodiesel production, and enzymes that oxidize this compound have been long sought after. The recently described alcohol oxidase from the white-rot basidiomycete ...Phanerochaete chrysosporium (PcAOX) was reported to feature very mild activity on glycerol. Here, we describe the comprehensive structural and biochemical characterization of this enzyme. PcAOX was expressed in Escherichia coli in high yields and displayed high thermostability. Steady-state kinetics revealed that PcAOX is highly active toward methanol, ethanol, and 1-propanol (k cat = 18, 19, and 11 s–1, respectively), but showed very limited activity toward glycerol (k obs = 0.2 s–1 at 2 M substrate). The crystal structure of the homo-octameric PcAOX was determined at a resolution of 2.6 Å. The catalytic center is a remarkable solvent-inaccessible cavity located at the re side of the flavin cofactor. Its small size explains the observed preference for methanol and ethanol as best substrates. These findings led us to design several cavity-enlarging mutants with significantly improved activity toward glycerol. Among them, the F101S variant had a high k cat value of 3 s–1, retaining a high degree of thermostability. The crystal structure of F101S PcAOX was solved, confirming the site of mutation and the larger substrate-binding pocket. Our data demonstrate that PcAOX is a very promising enzyme for glycerol biotransformation.
Multitarget directed ligands (MTDLs) represent a promising frontier in tackling the complexity of multifactorial pathologies. The synergistic inhibition of monoamine oxidase B (MAO B) and ...acetylcholinesterase (AChE) is believed to provide a potentiated effect in the treatment of Alzheimer’s disease. Among previously reported micromolar or sub-micromolar coumarin-bearing dual inhibitors, compound 1 returned a tight-binding inhibition of MAO B (K i = 4.5 μM) and a +5.5 °C increase in the enzyme T m value. Indeed, the X-ray crystal structure revealed that binding of 1 produces unforeseen conformational changes at the MAO B entrance cavity. Interestingly, 1 showed great shape complementarity with the AChE enzymatic gorge, being deeply buried from the catalytic anionic subsite (CAS) to the peripheral anionic subsite (PAS) and causing significant structural changes in the active site. These findings provide structural templates for further development of dual MAO B and AChE inhibitors.
Lysine-specific histone demethylase 1 (LSD1) is a very recently discovered enzyme which specifically removes methyl groups from Lys4 of histone 3. We have addressed the functional properties of the ...protein demonstrating that histone demethylation involves the flavin-catalysed oxidation of the methylated lysine. The nature of the substrate that acts as the electron acceptor required to complete the catalytic cycle was investigated. LSD1 converts oxygen to hydrogen peroxide although this reactivity is not as pronounced as that of other flavin-dependent oxidases. Our findings raise the possibility that in vivo LSD1 might not necessarily function as an oxidase, but it might use alternative electron acceptors.
The structural elucidations of human monoamine oxidases A and B (MAO-A and -B) have provided novel insights into their similarities and differences. Although the enzymes exhibit ∼70% sequence ...identities, highly conserved chain folds, and are structurally identical in their flavin adenine dinucleotide (FAD)-binding sites, they differ considerably in the structures of their active sites opposite the flavin cofactor. MAO-A has a monopartite cavity of ∼550 ų, and MAO-B exhibits a bipartite cavity structure with an entrance cavity of 290 ų and a substrate cavity of ∼400 ų. Ile199 functions as a conformational "gate" separating the two cavities. Both enzymes are anchored to the outer mitochondrial membrane via C-terminal helical tails. Loop structures are found at the entrances to their active sites at the membrane surface. Although the crystal structure of human MAO-A is monomeric while MAO-B is dimeric, both enzymes are dimeric in their membrane-bound forms. Dimerization may be important for the favorable orientation of the resultant protein dipole moment toward the anionic membrane surface.
Cardiac senescence is a typical chronic frailty condition in the elderly population, and cellular aging is often associated with oxidative stress. The mitochondrial-membrane flavoenzyme monoamine ...oxidase A (MAO A) catalyzes the oxidative deamination of neurotransmitters, and its expression increases in aged hearts. We produced recombinant human MAO A variants at Lys305 that play a key role in O2 reactivity leading to H2O2 production. The K305Q variant is as active as the wild-type enzyme, whereas K305M and K305S have 200-fold and 100-fold lower k cat values and similar K m. Under anaerobic conditions, K305M MAO A was normally reduced by substrate, whereas reoxidation by O2 was much slower but could be accomplished by quinone electron acceptors. When overexpressed in cardiomyoblasts by adenoviral vectors, the K305M variant showed enzymatic turnover similar to that of the wild-type but displayed decreased ROS levels and senescence markers. These results might translate into pharmacological treatments as MAO inhibitors may attenuate cardiomyocytes aging.
Various flavoprotein oxidases were recently shown to oxidize primary thiols. Herein, this reactivity is extended to sec‐thiols by using structure‐guided engineering of 5‐(hydroxymethyl)furfural ...oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of racemic sec‐thiols, thus yielding the corresponding thioketones and nonreacted R‐configured thiols with excellent enantioselectivities (E≥200). The engineering strategy applied went beyond the classic approach of replacing bulky amino acid residues with smaller ones, as the active site was additionally enlarged by a newly introduced Thr residue. This residue established a hydrogen‐bonding interaction with the substrates, as verified in the crystal structure of the variant. These strategies unlocked HMFO variants for the enantioselective oxidation of a range of sec‐thiols.
Engineered to fit: Rationally designed variants of 5‐(hydroxymethyl)furfural (HMF) oxidase catalyze the enantioselective oxidation of 1‐phenylethanethiols, thus achieving their kinetic resolution with unprecedented selectivities. The crystal structure of one of the variants explains this unique finding.
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