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Andrea Mattevi

Publications and source records attributed to Andrea Mattevi.

32 records · Page 2Linked to original sources
PubMed2004

Crystal structures of monoamine oxidase B in complex with four inhibitors of the N-propargylaminoindan class.

Monoamine oxidase B (MAO B) is an outer mitochondrial membrane enzyme that catalyzes the oxidation of arylalkylamine neurotransmitters. The crystal structures of MAO B in complex with four of the N-propargylaminoindan class of MAO covalent inhibitors (rasagiline, N-propargyl-1(S)-aminoindan, 6-hydroxy-N-propargyl-1(R)-aminoindan, and N-methyl-N-propargyl-1(R)-aminoindan) have been determined at a resolution of better than 2.1 A. Rasagiline, 6-hydroxy-N-propargyl-1(R)-aminoindan, and N-methyl-N-propargyl-1(R)-aminoindan adopt essentially the same conformation with the extended propargyl chain covalently bound to the flavin and the indan ring located in the rear of the substrate cavity. N-Propargyl-1(S)-aminoindan binds with the indan ring in a flipped conformation with respect to the other inhibitors, which causes a slight movement of the Tyr326 side chain. Four ordered water molecules are an integral part of the active site and establish H-bond interactions to the inhibitor atoms. These structural studies may guide future drug design to improve selectivity and efficacy by introducing appropriate substituents on the rasagiline molecular scaffold.

Alkynes↗
PubMed2004

Inactivation of purified human recombinant monoamine oxidases A and B by rasagiline and its analogues.

The inactivation of purified human recombinant monoamine oxidases (MAO) A and B by rasagiline [N-propargyl-1(R)-aminoindan] and four of its analogues [N-propargyl-1(S)-aminoindan (S-PAI), 6-hydroxy-N-propargyl-1(R)-aminoindan (R-HPAI), N-methyl-N-propargyl-1(R)-aminoindan (R-MPAI), and 6-(N-methyl-N-ethyl carbamoyloxy)-N-propargyl-1(R)-aminoindan (R-CPAI)] has been investigated. All compounds tested, with the exception of R-CPAI, form stoichiometric N(5) flavocyanine adducts with the FAD moiety of either enzyme. No H(2)O(2) is produced during either MAO A or MAO B inactivation, which demonstrates that covalent addition occurs in a single turnover. Rasagiline has the highest specificity for MAO B, as demonstrated by a 100-fold higher inhibition potency (k(inact)/K(i)) compared to MAO A, with the remaining compounds exhibiting lower isozyme specificities. MAO B and MAO A are more selective for the R-enantiomer (rasagiline) compared to the S-enantiomer (S-PAI) by 2500-fold and 17-fold, respectively. Differences in UV/vis and CD spectral data of the complexes of the studied compounds with both MAO A and MAO B are interpreted in light of crystallographic data of complexes of MAO B with rasagiline and its analogues (Binda, C.; et al. J. Med. Chem. 2004, 47, 1767-1774.

Alkynes↗
PubMed2004

The FAD binding sites of human monoamine oxidases A and B.

The structural details of the interactions of the covalent 8alpha-S-cysteinyl-FAD with the protein moiety in monoamine oxidase B (MAO B) based on the MAO B crystal structure are described. The dinucleotide is bound to the protein in an extended conformation with the majority of the bonds to the protein identified as hydrogen bonds with amino acid side chains, amide bonds, and water molecules. Since those amino acids interacting with the FAD are conserved in monoamine oxidase A (MAO A), it is proposed that the FAD binding site in MAO A is quite similar to that in MAO B. The redox-active isoalloxazine ring is buried in the protein without direct access to bulk solvent. An electrostatic interaction is observed between the anionic pyrophosphate moiety and Arg42. The normally flat oxidized flavin ring is in a bent, puckered conformation in the MAO B binding site which is suggested to contribute to its reactivity in catalysis. This structural information is then used to explain previous studies on flavin analog incorporation into either MAO B or into MAO A.

Animals↗
PubMed2003

Structural studies on flavin reductase PheA2 reveal binding of NAD in an unusual folded conformation and support novel mechanism of action.

The catabolism of toxic phenols in the thermophilic organism Bacillus thermoglucosidasius A7 is initiated by a two-component enzyme system. The smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols. We have determined the x-ray structure of PheA2 containing a bound FAD cofactor (2.2 A), which is the first structure of a member of this flavin reductase family. We have also determined the x-ray structure of reduced holo-PheA2 in complex with oxidized NAD (2.1 A). PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded beta-sheet and a capping alpha-helix. The tightly bound FAD prosthetic group (K(d) = 10 nm) binds near the dimer interface, and the re face of the FAD isoalloxazine ring is fully exposed to solvent. The addition of NADH to crystalline PheA2 reduced the flavin cofactor, and the NAD product was bound in a wide solvent-accessible groove adopting an unusual folded conformation with ring stacking. This is the first observation of an enzyme that is very likely to react with a folded compact pyridine nucleotide. The PheA2 crystallographic models strongly suggest that reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product. Nanoflow electrospray mass spectrometry data indeed showed that PheA2 is able to bind one FAD cofactor and one FAD substrate. In conclusion, the structural data provide evidence that PheA2 contains a dual binding cleft for NADH and FAD substrate, which alternate during catalysis.

Amino Acid Sequence↗
PubMed2003

Polystyrene microbridges used in sitting-drop crystallization release 1,4-diphenyl-2-butene, a novel inhibitor of human MAO B.

In the course of protein-structure determinations of the membrane-bound enzyme monoamine oxidase B (MAO B) by X-ray crystallography, a compound was found in the active site of the enzyme that consists of two phenyl rings separated by four C atoms. This compound was identified by chromatography and by mass spectrometry to be 1,4-diphenyl-2-butene and found to be a component of the polystyrene microbridges that are used in protein crystallization. This compound is present at a level of approximately 0.3 mg ( approximately 1.5 micro mol) per microbridge and functions as a competitive inhibitor of MAO B with a K(i) of 35 micro M. The presence of detergents in the crystallization solutions facilitates the extraction of this compound from the polymer medium.

Alkenes↗
PubMed2003

Insights into the mode of inhibition of human mitochondrial monoamine oxidase B from high-resolution crystal structures.

Monoamine oxidase B (MAO-B) is an outer mitochondrial membrane-bound enzyme that catalyzes the oxidative deamination of arylalkylamine neurotransmitters and has been a target for a number of clinically used drug inhibitors. The 1.7-A structure of the reversible isatin-MAO-B complex has been determined; it forms a basis for the interpretation of the enzyme's structure when bound to either reversible or irreversible inhibitors. 1,4-Diphenyl-2-butene is found to be a reversible MAO-B inhibitor, which occupies both the entrance and substrate cavity space in the enzyme. Comparison of these two structures identifies Ile-199 as a "gate" between the two cavities. Rotation of the side chain allows for either separation or fusion of the two cavities. Inhibition of the enzyme with N-(2-aminoethyl)-p-chlorobenzamide results in the formation of a covalent N(5) flavin adduct with the phenyl ring of the inhibitor occupying a position in the catalytic site overlapping that of isatin. Inhibition of MAO-B with the clinically used trans-2-phenylcyclopropylamine results in the formation of a covalent C(4a) flavin adduct with an opened cyclopropyl ring and the phenyl ring in a parallel orientation to the flavin. The peptide bond between the flavin-substituted Cys-397 and Tyr-398 is in a cis conformation, which allows the proper orientation of the phenolic ring of Tyr-398 in the active site. The flavin ring exists in a twisted nonplanar conformation, which is observed in the oxidized form as well as in both the N(5) and the C(4a) adducts. An immobile water molecule is H-bonded to Lys-296 and to the N(5) of the flavin as observed in other flavin-dependent amine oxidases. The active site cavities are highly apolar; however, hydrophilic areas exist near the flavin and direct the amine moiety of the substrate for binding and catalysis. Small conformational changes are observed on comparison of the different inhibitor-enzyme complexes. Future MAO-B drug design will need to consider "induced fit" contributions as an element in ligand-enzyme interactions.

Catalytic Domain↗
PubMed2003

The active conformation of glutamate synthase and its binding to ferredoxin.

Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. The plant-type ferredoxin-dependent GltS and the functionally homologous alpha subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family of amidotransferases. The enzymes function through the channeling of ammonia from the N-terminal amidotransferase domain to the FMN-binding domain. Here, we report the X-ray structure of the Synechocystis ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct active sites solved using a new crystal form. The covalent Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis. Moreover, we determined a high resolution structure of the GltS:2-oxoglutarate complex. These structures represent the enzyme in the active conformation. By comparing these structures with that of GltS alpha subunit and of related enzymes we propose a mechanism for enzyme self-regulation and ammonia channeling between the active sites. X-ray small-angle scattering experiments were performed on solutions containing GltS and its physiological electron donor ferredoxin (Fd). Using the structure of GltS and the newly determined crystal structure of Synechocystis Fd, the scattering experiments clearly showed that GltS forms an equimolar (1:1) complex with Fd. A fundamental consequence of this result is that two Fd molecules bind consecutively to Fd-GltS to yield the reduced FMN cofactor during catalysis.

Crystallography, X-Ray↗
PubMed2002

A covalent modification of NADP+ revealed by the atomic resolution structure of FprA, a Mycobacterium tuberculosis oxidoreductase.

FprA is a mycobacterial oxidoreductase that catalyzes the transfer of reducing equivalents from NADPH to a protein acceptor. We determined the atomic resolution structure of FprA in the oxidized (1.05 A resolution) and NADPH-reduced (1.25 A resolution) forms. The comparison of these FprA structures with that of bovine adrenodoxin reductase showed no significant overall differences. Hence, these enzymes, which belong to the structural family of the disulfide oxidoreductases, are structurally conserved in very distant organisms such as mycobacteria and mammals. Despite the conservation of the overall fold, the details of the active site of FprA show some peculiar features. In the oxidized enzyme complex, the bound NADP+ exhibits a covalent modification, which has been identified as an oxygen atom linked through a carbonylic bond to the reactive C4 atom of the nicotinamide ring. Mass spectrometry has confirmed this assignment. This NADP+ derivative is likely to form by oxidation of the NADP+ adduct resulting from nucleophilic attack by an active-site water molecule. A Glu-His pair is well positioned to activate the attacking water through a mechanism analogous to that of the catalytic triad in serine proteases. The NADP+ nicotinamide ring exhibits the unusual cis conformation, which may favor derivative formation. The physiological significance of this reaction is presently unknown. However, it could assist with drug-design studies in that the modified NADP+ could serve as a lead compound for the development of specific inhibitors.

Amino Acid Sequence↗
PubMed2002

Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit.

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.

Amino Acid Oxidoreductases↗
PubMed2002

Structural studies on the synchronization of catalytic centers in glutamate synthase.

The complex iron-sulfur flavoprotein glutamate synthase (GltS) plays a prominent role in ammonia assimilation in bacteria, yeasts, and plants. GltS catalyzes the formation of two molecules of l-glutamate from 2-oxoglutarate and l-glutamine via intramolecular channeling of ammonia. GltS has the impressive ability of synchronizing its distinct catalytic centers to avoid wasteful consumption of l-glutamine. We have determined the crystal structure of the ferredoxin-dependent GltS in several ligation and redox states. The structures reveal the crucial elements in the synchronization between the glutaminase site and the 2-iminoglutarate reduction site. The structural data combined with the catalytic properties of GltS indicate that binding of ferredoxin and 2-oxoglutarate to the FMN-binding domain of GltS induce a conformational change in the loop connecting the two catalytic centers. The rearrangement induces a shift in the catalytic elements of the amidotransferase domain, such that it becomes activated. This machinery, over a distance of more than 30 A, controls the ability of the enzyme to bind and hydrolyze the ammonia-donating substrate l-glutamine.

Amino Acid Oxidoreductases↗
PubMed2002

Structure and function of human erythrocyte pyruvate kinase. Molecular basis of nonspherocytic hemolytic anemia.

Deficiency of human erythrocyte isozyme (RPK) is, together with glucose-6-phosphate dehydrogenase deficiency, the most common cause of the nonspherocytic hemolytic anemia. To provide a molecular framework to the disease, we have solved the 2.7 A resolution crystal structure of human RPK in complex with fructose 1,6-bisphosphate, the allosteric activator, and phosphoglycolate, a substrate analogue, and we have functionally and structurally characterized eight mutants (G332S, G364D, T384M, D390N, R479H, R486W, R504L, and R532W) found in RPK-deficient patients. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. The mutations affect to a different extent thermostability, catalytic efficiency, and regulatory properties. These studies are the first to correlate the clinical symptoms with the molecular properties of the mutant enzymes. Mutations greatly impairing thermostability and/or activity are associated with severe anemia. Some mutant proteins exhibit moderate changes in the kinetic parameters, which are sufficient to cause mild to severe anemia, underlining the crucial role of RPK for erythrocyte metabolism. Prediction of the effects of mutations is difficult because there is no relation between the nature and location of the replaced amino acid and the type of molecular perturbation. Characterization of mutant proteins may serve as a valuable tool to assist with diagnosis and genetic counseling.

Allosteric Regulation↗
PubMed2002

Structure of FAD-bound L-aspartate oxidase: insight into substrate specificity and catalysis.

L-Aspartate oxidase (Laspo) catalyzes the conversion of L-Asp to iminoaspartate, the first step in the de novo biosynthesis of NAD(+). This bacterial pathway represents a potential drug target since it is absent in mammals. The Laspo R386L mutant was crystallized in the FAD-bound catalytically competent form and its three-dimensional structure determined at 2.5 A resolution in both the native state and in complex with succinate. Comparison of the R386L holoprotein with the wild-type apoenzyme [Mattevi, A., Tedeschi, G., Bacchella, L., Coda, A., Negri, A., and Ronchi, S. (1999) Structure 7, 745-756] reveals that cofactor incorporation leads to the ordering of two polypeptide segments (residues 44-53 and 104-141) and to a 27 degree rotation of the capping domain. This motion results in the formation of the active site cavity, located at the interface between the capping domain and the FAD-binding domain. The structure of the succinate complex indicates that the cavity surface is decorated by two clusters of H-bond donors that anchor the ligand carboxylates. Moreover, Glu121, which is strictly conserved among Laspo sequences, is positioned to interact with the L-Asp alpha-amino group. The architecture of the active site of the Laspo holoenzyme is remarkably similar to that of respiratory fumarate reductases, providing strong evidence for a common mechanism of catalysis in Laspo and flavoproteins of the succinate dehydrogenase/fumarate reductase family. This implies that Laspo is mechanistically distinct from other flavin-dependent amino acid oxidases, such as the prototypical D-amino acid oxidase.

Amino Acid Oxidoreductases↗
PubMed2002

Structure of human monoamine oxidase B, a drug target for the treatment of neurological disorders.

Monoamine oxidase B (MAO B) is a mitochondrial outermembrane flavoenzyme that is a well-known target for antidepressant and neuroprotective drugs. We determined the structure of the human enzyme to 3 A resolution. The enzyme binds to the membrane through a C-terminal transmembrane helix and apolar loops located at various positions in the sequence. The electron density shows that pargyline, an analog of the clinically used MAO B inhibitor, deprenyl, binds covalently to the flavin N5 atom. The active site of MAO B consists of a 420 A(3)-hydrophobic substrate cavity interconnected to an entrance cavity of 290 A(3). The recognition site for the substrate amino group is an aromatic cage formed by Tyr 398 and Tyr 435. The structure provides a framework for probing the catalytic mechanism, understanding the differences between the B- and A-monoamine oxidase isoforms and designing specific inhibitors.

Amino Acid Sequence↗