Crystallization of Human Monoamine Oxidase B.

The interest in monoamine oxidases A and B (MAO A and B) is due to their central role in regulating the balance of neurotransmitters, both in the central nervous system and in peripheral organs. As validated drug targets for depression and Parkinson's disease, the elucidation of their crystal structures was an essential step to guide drug design investigations. The development of the heterologous expression system of MAO B in Pichia pastoris and the identification of the detergent, buffer, and precipitant conditions allowed to determine the first crystal structure of human MAO B in 2002.

Purification of Recombinant Eukaryotic MAO A and MAO B Utilizing the Pichia pastoris Expression System.

Procedures are described for the heterologous expression and purification of the mitochondrial-bound enzymes human and rat monoamine oxidases A and B and zebrafish MAO in the yeast Pichia pastoris. Enzyme expression is under control of a methanol oxidase promoter and similar procedures have been developed for the preparation of membrane particles and detergent solubilization of the functional enzymes. Similarities and differences are described in the procedures for purification of the respective enzymes using standard column chromatographic techniques to provide enzyme yields in the range of 100-300 mg from 1 L of cell culture.

Purification of MAO A and MAO B from Mammalian Tissue Sources.

Procedures are described for the purification of the mitochondrial-bound enzymes human and bovine monoamine oxidases A and B (MAO A and B) from placental and liver tissue sources, respectively. Enzyme purification follows isolation of the mitochondria and preparation of outer membrane particles. The membrane-bound enzymes are solubilized by treatment of membranes with phospholipases and detergent extraction.

Rational Redesign of Monoamine Oxidase A into a Dehydrogenase to Probe ROS in Cardiac Aging.

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 O reactivity leading to HO production.

Monoamine Oxidases.

Monoamine oxidases A and B (MAO A and B) are mammalian flavoenzymes bound to the outer mitochondrial membrane. They were discovered almost a century ago and they have been the subject of many biochemical, structural and pharmacological investigations due to their central role in neurotransmitter metabolism. Currently, the treatment of Parkinson's disease involves the use of selective MAO B inhibitors such as rasagiline and safinamide.

Monoamine Oxidases, Oxidative Stress, and Altered Mitochondrial Dynamics in Cardiac Ageing.

The advances in healthcare over the past several decades have resulted in populations now living longer. With this increase in longevity, a wider prevalence of cardiovascular diseases is more common and known to be a major factor in rising healthcare costs. A wealth of scientific evidence has implicated cell senescence as an important component in the etiology of these age-dependent pathologies.

Why p-OMe- and p-Cl-β-Methylphenethylamines Display Distinct Activities upon MAO-B Binding.

Despite their structural and chemical commonalities, p-chloro-β-methylphenethylamine and p-methoxy-β-methylphenethylamine display distinct inhibitory and substrate activities upon MAO-B binding. Density Functional Theory (DFT) quantum chemical calculations reveal that β-methylation and para-substitution underpin the observed activities sustained by calculated transition state energy barriers, attained conformations and key differences in their interactions in the enzyme's substrate binding site. Although both compounds meet substrate requirements, it is clear that β-methylation along with the physicochemical features of the para-substituents on the aromatic ring determine the activity of these compounds upon binding to the MAO B-isoform.

Design and synthesis of novel chalcones as potent selective monoamine oxidase-B inhibitors.

A novel series of substituted chalcones were designed and synthesized to be evaluated as selective human MAO-B inhibitors. A combination of either methylsulfonyl or trifluoromethyl substituents on the aromatic ketone moiety with a benzodioxol ring on the other end of the chalcone scaffold was investigated. The compounds were tested for their inhibitory activities on both human MAO-A and B.

Reversible and irreversible small molecule inhibitors of monoamine oxidase B (MAO-B) investigated by biophysical techniques.

Monoamine oxidase B (MAO-B) plays a key role in the metabolism of dopamine, a neurotransmitter critical for the maintenance of cognitive function. Consequently, MAO-B is an important therapeutic target for disorders characterized by a decline in dopaminergic neurotransmission, including Parkinson's disease (PD). An emerging strategy in drug discovery is to utilize the biophysical approaches of thermal shift and isothermal titration calorimetry (ITC) to gain insight into binding modality and identify thermodynamically privileged chemical scaffolds.

Truncated FAD synthetase for direct biocatalytic conversion of riboflavin and analogs to their corresponding flavin mononucleotides.

The preparation of flavin mononucleotide (FMN) and FMN analogs from their corresponding riboflavin precursors is traditionally performed in a two-step procedure. After initial enzymatic conversion of riboflavin to flavin adenine dinucleotide (FAD) by a bifunctional FAD synthetase, the adenyl moiety of FAD is hydrolyzed with snake venom phosphodiesterase to yield FMN. To simplify the protocol, we have engineered the FAD synthetase from Corynebacterium ammoniagenes by deleting its N-terminal adenylation domain.

Hydrogen peroxide produced by mitochondrial monoamine oxidase catalysis: biological implications.

The biological roles of mitochondrial-produced reactive oxygen species continue to receive intensive investigation since one of the products (H₂O₂) has important cellular signaling roles as well as contributing to apoptotic responses. In general, the source of mitochondrial reactive oxygen species is thought to be the superoxide anion produced from Complex I and Complex III components of the electron transport chain. Superoxide anion readily dismutates to H₂O₂ with subsequent transformation to the hydroxyl radical by Fenton chemistry.

Precursor of ether phospholipids is synthesized by a flavoenzyme through covalent catalysis.

The precursor of the essential ether phospholipids is synthesized by a peroxisomal enzyme that uses a flavin cofactor to catalyze a reaction that does not alter the redox state of the substrates. The enzyme crystal structure reveals a V-shaped active site with a narrow constriction in front of the prosthetic group. Mutations causing inborn ether phospholipid deficiency, a very severe genetic disease, target residues that are part of the catalytic center.

Structural basis for benzothiazinone-mediated killing of Mycobacterium tuberculosis.

The benzothiazinone BTZ043 is a tuberculosis drug candidate with nanomolar whole-cell activity. BTZ043 targets the DprE1 catalytic component of the essential enzyme decaprenylphosphoryl-β-D-ribofuranose-2'-epimerase, thus blocking biosynthesis of arabinans, vital components of mycobacterial cell walls. Crystal structures of DprE1, in its native form and in a complex with BTZ043, reveal formation of a semimercaptal adduct between the drug and an active-site cysteine, as well as contacts to a neighboring catalytic lysine residue.

Molecular Insights into Human Monoamine Oxidase B Inhibition by the Glitazone Anti-Diabetes Drugs.

The widely employed anti-diabetic drug pioglitazone (Actos) is shown to be a specific and reversible inhibitor of human monoamine oxidase B (MAO B). The crystal structure of the enzyme-inhibitor complex shows the R-enantiomer is bound with the thiazolidinedione ring near the flavin. The molecule occupies both substrate and entrance cavities of the active site establishing non-covalent interactions with the surrounding amino acids.

Lights and shadows on monoamine oxidase inhibition in neuroprotective pharmacological therapies.

Playing a pivotal role in the metabolism of neurotransmitters in the central nervous system, the mitochondrial enzymes monoamine oxidases A and B (MAO A and B) have been for long studied as drug targets for neurodegenerative and neurological diseases. MAO inhibitors (MAOIs) are clinically used to treat Parkinson's disease and depression by blocking the degradation of neuroactive catecholamines and providing a symptomatic relief in the patients. More recent is the idea that the neuroprotective effect of MAOIs may result from the prevention of oxidative stress produced by the MAO reaction rather than being simply related to the inhibition of neurotransmitters degradation.

The 'gating' residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition.

The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 Å(3) volume and MAO B contains a dipartite cavity structure with volumes of ~290 Å(3) (entrance cavity) and ~400 Å(3) (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala-Tyr326Ala mutant forms of MAO B were investigated.

Structural properties of human monoamine oxidases A and B.

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.

²H kinetic isotope effects and pH dependence of catalysis as mechanistic probes of rat monoamine oxidase A: comparisons with the human enzyme.

Monoamine oxidase A (MAO A) is a mitochondrial outer membrane-bound flavoenzyme important in the regulation of serotonin and dopamine levels. Because the rat is extensively used as an animal model in drug studies, it is important to understand how rat MAO A behaves in comparison with the more extensively studied human enzyme. For many reversible inhibitors, rat MAO A exhibits K(i) values similar to those of human MAO A.

Nitrogen kinetic isotope effects for the monoamine oxidase B-catalyzed oxidation of benzylamine and (1,1-(2)H2)benzylamine: nitrogen rehybridization and CH bond cleavage are not concerted.

Nitrogen kinetic isotope effects for the oxidation of benzylamine and (1,1-(2)H(2))benzylamine by recombinant human monoamine oxidase B show that cleavage of the CH bond is not concerted with rehybridization of the nitrogen atom.

Catalytic and inhibitor binding properties of zebrafish monoamine oxidase (zMAO): comparisons with human MAO A and MAO B.

A comparative investigation of substrate specificity and inhibitor binding properties of recombinant zebrafish (Danio rerio) monoamine oxidase (zMAO) with those of recombinant human monoamine oxidases A and B (hMAO A and hMAO B) is presented. zMAO oxidizes the neurotransmitter amines (serotonin, dopamine and tyramine) with k(cat) values that exceed those of hMAO A or of hMAO B. The enzyme is competitively inhibited by hMAO A selective reversible inhibitors with the exception of d-amphetamine where uncompetitive inhibition is exhibited.

Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis.

TEMPO-substituted pargyline analogues differentially inhibit recombinant human monoamine oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria, suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane [Upadhyay, A., and Edmondson, D. E.

Interactions of monoamine oxidases with the antiepileptic drug zonisamide: specificity of inhibition and structure of the human monoamine oxidase B complex.

The binding of zonisamide to purified, recombinant monoamine oxidases (MAOs) has been investigated. It is a competitive inhibitor of human MAO B (K(i) = 3.1 ± 0.

Potentiation of ligand binding through cooperative effects in monoamine oxidase B.

Crystallographic and biochemical studies have been employed to identify the binding site and mechanism for potentiation of imidazoline binding in human monoamine oxidase B (MAO B). 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) inhibits recombinant human MAO B with a K(i) of 8.3 ± 0.

Biochemical, structural, and biological evaluation of tranylcypromine derivatives as inhibitors of histone demethylases LSD1 and LSD2.

LSD1 and LSD2 histone demethylases are implicated in a number of physiological and pathological processes, ranging from tumorigenesis to herpes virus infection. A comprehensive structural, biochemical, and cellular study is presented here to probe the potential of these enzymes for epigenetic therapies. This approach employs tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors.