PubMed Health⌕ Search

Biomedical subjects

Andrea Mattevi

Publications and source records attributed to Andrea Mattevi.

At least 19 recordsLinked to original sources
PubMed2007

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase.

p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii is a two-component system consisting of a NADH-dependent FMN reductase and a monooxygenase (C2) that uses reduced FMN as substrate. The crystal structures of C2 in the ligand-free and substrate-bound forms reveal a preorganized pocket that binds reduced FMN without large conformational changes. The Phe-266 side chain swings out to provide the space for binding p-hydroxyphenylacetate that is oriented orthogonal to the flavin ring. The geometry of the substrate-binding site of C2 is significantly different from that of p-hydroxybenzoate hydroxylase, a single-component flavoenzyme that catalyzes a similar reaction. The C2 overall structure resembles the folding of medium-chain acyl-CoA dehydrogenase. An outstanding feature in the C2 structure is a cavity located in front of reduced FMN; it has a spherical shape with a 1.9-A radius and a 29-A3 volume and is interposed between the flavin C4a atom and the substrate atom to be hydroxylated. The shape and position of this cavity are perfectly fit for housing the oxygen atoms of the flavin C4a-hydroperoxide intermediate that is formed upon reaction of the C2-bound reduced flavin with molecular oxygen. The side chain of His-396 is predicted to act as a hydrogen-bond donor to the oxygen atoms of the intermediate. This architecture promotes the nucleophilic attack of the substrate onto the terminal oxygen of the hydroperoxyflavin. Comparative analysis with the structures of other flavoenzymes indicates that a distinctive feature of monooxygenases is the presence of specific cavities that encapsulate and stabilize the crucial hydroperoxyflavin intermediate.

Catalysis↗
PubMed2006

Crystallization and preliminary X-ray analysis of an alditol oxidase from Streptomyces coelicolor A3(2).

Alditol oxidase is a 45 kDa enzyme containing a covalently bound FAD cofactor. This oxidase efficiently oxidizes a range of alditols to the corresponding aldoses. Owing to its substrate range and regioselectivity, this enzyme is an interesting candidate for biotechnological applications. Crystals of alditol oxidase from Streptomyces coelicolor A3(2) were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.1 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 107, b = 68, c = 58 A, beta = 94 degrees. Crystals of seleno-L-methionine-labelled alditol oxidase were obtained after seeding the crystallization drops with native microcrystals and showed a diffraction limit of 2.4 A.

Alcohol Oxidoreductases↗
PubMed2006

New frontiers in structural flavoenzymology.

During the past few years, there have been exciting developments in the field of flavoenzymology. New flavoenzymes have been discovered that are implicated in a variety of biological processes, including cell signaling, chromatin remodeling and cell development. The structures of several of these new flavoenzymes have been described, as exemplified by crystallographic analyses of MICAL, histone demethylase LSD1 and tryptophan dehalogenase. In addition, new structural information has revealed the evolutionary and mechanistic complexity of the enzymes of the riboflavin biosynthetic pathway. The integration of the enzymology data with crystallographic studies at atomic resolution is resulting in unprecedented insight into the chemical and geometric properties underlying flavoenzyme function.

Catalysis↗
PubMed2006

A highly specific mechanism of histone H3-K4 recognition by histone demethylase LSD1.

Human lysine-specific demethylase (LSD1) is a chromatin-modifying enzyme that specifically removes methyl groups from mono- and dimethylated Lys4 of histone H3 (H3-K4). We used a combination of in vivo and in vitro experiments to characterize the substrate specificity and recognition by LSD1. Biochemical assays on histone peptides show that essentially all epigenetic modifications on the 21 N-terminal amino acids of histone H3 cause a significant reduction in enzymatic activity. Replacement of Lys4 with Arg greatly enhances binding affinity, and a histone peptide incorporating this mutation has a strong inhibitory power. Conversely, a peptide bearing a trimethylated Lys4 is only a weak inhibitor of the enzyme. Rapid kinetics measurements evidence that the enzyme is efficiently reoxidized by molecular oxygen with a second-order rate constant of 9.6x10(3) M-1 s-1, and that the presence of the reaction product does not greatly influence the rate of flavin reoxidation. In vivo experiments provide a correlation between the in vitro inhibitory properties of the tested peptides and their ability of affecting endogenous LSD1 activity. Our results show that epigenetic modifications on histone H3 need to be removed before Lys4 demethylation can efficiently occur. The complex formed by LSD1 with histone deacetylases 1/2 may function as a "double-blade razor" that first eliminates the acetyl groups from acetylated Lys residues and then removes the methyl group from Lys4. We suggest that after H3-K4 demethylation, LSD1 recruits the forthcoming chromatin remodelers leading to the introduction of gene repression marks.

Cell Line↗
PubMed2006

Role of the His57-Glu214 ionic couple located in the active site of Mycobacterium tuberculosis FprA.

Mycobacterium tuberculosis FprA is a NADPH-ferredoxin reductase, functionally and structurally similar to the mammalian adrenodoxin reductase. It is presumably involved in supplying electrons to one or more of the pathogen's cytochrome P450s through reduced ferredoxins. It has been proposed on the basis of crystallographic data (Bossi, R. T., et al. (2002) Biochemistry 41, 8807-8818) that the highly conserved His57 and Glu214 whose side chains are H-bonded are involved in catalysis. Both residues were individually changed to nonionizable amino acyl residues through site-directed mutagenesis. Steady-state kinetics showed that the role of Glu214 in catalysis is negligible. On the contrary, the substitutions of His57 markedly impaired the catalytic efficiency of FprA for ferredoxin in the physiological reaction. Furthemore, they decreased the k(cat)/K(m) value for NADPH in the ferricyanide reduction. Rapid-reaction (stopped-flow) kinetic analysis of the isolated reductive half-reaction of wild-type and His57Gln forms of FprA with NADPH and NADH allowed a detailed description of the mechanism of enzyme-bound FAD reduction, with the identification of the intermediates involved. The His57Gln mutation caused a 6-fold decrease in the rate of hydride transfer from either NADPH or NADH to the enzyme-bound FAD cofactor. The 3D structure of FprA-H57Q, obtained at 1.8 A resolution, explains the inefficient hydride transfer of the mutant in terms of a suboptimal geometry of the nicotinamide-isoalloxazine interaction in the active site. These data demonstrate the role of His57 in the correct binding of NADPH to FprA for the subsequent steps of the catalytic cycle to proceed at a high rate.

Amino Acid Sequence↗
PubMed2006

Functional role of the "aromatic cage" in human monoamine oxidase B: structures and catalytic properties of Tyr435 mutant proteins.

Current structural results of several flavin-dependent amine oxidizing enzymes including human monoamine oxidases A and B (MAO A and MAO B) show aromatic amino acid residues oriented approximately perpendicular to the flavin ring, suggesting a functional role in catalysis. In the case of human MAO B, two tyrosyl residues (Y398 and Y435) are found in the substrate binding site on the re face of the covalent flavin ring [Binda et al. (2002) J. Biol. Chem. 277, 23973-23976]. To probe the functional significance of this structure, Tyr435 in MAO B was mutated with the amino acids Phe, His, Leu, or Trp, the mutant proteins expressed in Pichia pastoris, and purified to homogeneity. Each mutant protein contains covalent FAD and exhibits a high level of catalytic functionality. No major alterations in active site structures are detected on comparison of their respective crystal structures with that of WT enzyme. The relative k(cat)/K(m) values for each mutant enzyme show Y435 > Y435F = Y435L = Y435H > Y435W. A similar behavior is also observed with the membrane-bound forms of MAO A and MAO B (MAO A Y444 mutant enzymes are found to be unstable on membrane extraction). p-Nitrobenzylamine is found to be a poor substrate while p-nitrophenethylamine is found to be a good substrate for all WT and mutant forms of MAO B. Analysis of these kinetic and structural data suggests the function of the "aromatic cage" in MAO to include a steric role in substrate binding and access to the flavin coenzyme and to increase the nucleophilicity of the substrate amine moiety. These results are consistent with a proposed polar nucleophilic mechanism for catalytic amine oxidation.

Amines↗
PubMed2006

To be or not to be an oxidase: challenging the oxygen reactivity of flavoenzymes.

Flavin-dependent enzymes catalyse a wide range of reactions and, thereby, facilitate a variety of cellular processes. Among the properties that equip flavoenzymes with this chemical versatility is their reactivity towards oxygen, which shows huge variation among flavoproteins. A survey of known 3D structures of flavin-dependent oxidases and dehydrogenases and the correlation with their functional properties indicates that there are no structural rules that enable prediction of whether or how a flavoenzyme reacts with oxygen. Combinations of subtle factors such as dipole pre-organization, charge distribution, dynamics and solvation in the active centre determine the balance of interactions that control oxygen reactivity. The chemical basis of oxygen reactivity remains a puzzling problem and represents one of the challenging questions in modern flavoenzymology.

Enzymes↗
PubMed2006

A monotopic membrane protein goes solo.

Carnitine palmitoyltransferases (CPTs) are part of the enzymatic system that imports fatty acids into mitochondria. The crystal structure of rat CPT-2 by Rufer et al. (2006) (this issue of Structure) reveals a Y-shaped tunnel for binding the CoA and acyl-carnitine substrates and a hydrophobic insert mediating membrane association.

Animals↗
PubMed2005

Binding of rasagiline-related inhibitors to human monoamine oxidases: a kinetic and crystallographic analysis.

Monoamine oxidases A and B (MAO A and B) catalyze the degradation of neurotransmitters and represent drug targets for the treatment of neurodegenerative disorders. Rasagiline is an irreversible, MAO B-selective inhibitor that has been approved as a novel anti-Parkinson's drug. In this study, we investigate the inhibition of recombinant human MAO A and MAO B by several rasagiline analogues. Different substituents added onto the rasagiline scaffold alter the binding affinity depending on the position on the aminoindan ring and on the size of the substituent. Compounds with a hydroxyl group on either the C4 or the C6 atom inhibit both isozymes, whereas a bulkier substituent such as a carbamate is tolerated only at the C4 position. The 1.7 A crystal structure of MAO B in complex with 4-(N-methyl-N-ethyl-carbamoyloxy)-N-methyl-N-propargyl-1(R)-aminoindan shows that the binding mode is similar to that of rasagiline with the carbamate moiety occupying the entrance cavity space. 1(R)-Aminoindan, the major metabolic product of rasagiline, and its analogues reversibly inhibit both MAO A and MAO B. The crystal structure of N-methyl-1(R)-aminoindan bound to MAO B shows that its aminoindan ring adopts a different orientation compared to that of rasagiline.

Crystallography, X-Ray↗
PubMed2005

Human histone demethylase LSD1 reads the histone code.

Human histone demethylase LSD1 is a flavin-dependent amine oxidase that catalyzes the specific removal of methyl groups from mono- and dimethylated Lys4 of histone H3. The N-terminal tail of H3 is subject to various covalent modifications, and a fundamental question in LSD1 biology is how these epigenetic marks affect the demethylase activity. We show that LSD1 does not have a strong preference for mono- or dimethylated Lys4 of H3. Substrate recognition is not confined to the residues neighboring Lys4, but it requires a sufficiently long peptide segment consisting of the N-terminal 20 amino acids of H3. Electrostatic interactions are an important factor in protein-substrate recognition, as indicated by the high sensitivity of Km to ionic strength. We have probed LSD1 for its ability to demethylate Lys4 in presence of a second modification on the same peptide substrate. Methylation of Lys9 does not affect enzyme catalysis. Conversely, Lys9 acetylation causes an almost 6-fold increase in the Km value, whereas phosphorylation of Ser10 totally abolishes activity. LSD1 is inhibited by a demethylated peptide with an inhibition constant of 1.8 microM, suggesting that LSD1 can bind to H3 independently of Lys4 methylation. LSD1 is a chromatin-modifying enzyme, which is able to read different epigenetic marks on the histone N-terminal tail and can serve as a docking module for the stabilization of the associated corepressor complex(es) on chromatin.

Amino Acid Sequence↗
PubMed2005

Three-dimensional structure of human monoamine oxidase A (MAO A): relation to the structures of rat MAO A and human MAO B.

The three-dimensional structure of recombinant human monoamine oxidase A (hMAO A) as its clorgyline-inhibited adduct is described. Although the chain-fold of hMAO A is similar to that of rat MAO A and human MAO B (hMAO B), hMAO A is unique in that it crystallizes as a monomer and exhibits the solution hydrodynamic behavior of a monomeric form rather than the dimeric form of hMAO B and rat MAO A. hMAO A's active site consists of a single hydrophobic cavity of approximately 550 A3, which is smaller than that determined from the structure of deprenyl-inhibited hMAO B (approximately 700 A3) but larger than that of rat MAO A (approximately 450 A3). An important component of the active site structure of hMAO A is the loop conformation of residues 210-216, which differs from that of hMAO B and rat MAO A. The origin of this structural alteration is suggested to result from long-range interactions in the monomeric form of the enzyme. In addition to serving as a basis for the development of hMAO A specific inhibitors, these data support the proposal that hMAO A involves a change from the dimeric to the monomeric form through a Glu-151 --> Lys mutation that is specific of hMAO A [Andrès, A. M., Soldevila, M., Navarro, A., Kidd, K. K., Oliva, B. & Bertranpetit, J. (2004) Hum. Genet. 115, 377-386]. These considerations put into question the use of MAO A from nonhuman sources in drug development for use in humans.

Animals↗
PubMed2005

Histone demethylation catalysed by LSD1 is a flavin-dependent oxidative process.

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.

Catalysis↗
PubMed2005

Demonstration of isoleucine 199 as a structural determinant for the selective inhibition of human monoamine oxidase B by specific reversible inhibitors.

Several reversible inhibitors selective for human monoamine oxidase B (MAO B) that do not inhibit MAO A have been described in the literature. The following compounds: 8-(3-chlorostyryl)caffeine, 1,4-diphenyl-2-butene, and trans,trans-farnesol are shown to inhibit competitively human, horse, rat, and mouse MAO B with K(i) values in the low micromolar range but are without effect on either bovine or sheep MAO B or human MAO A. In contrast, the reversible competitive inhibitor isatin binds to all known MAO B and MAO A with similar affinities. Sequence alignments and the crystal structures of human MAO B in complex with 1,4-diphenyl-2-butene or with trans,trans-farnesol provide molecular insights into these specificities. These inhibitors span the substrate and entrance cavities with the side chain of Ile-199 rotated out of its normal conformation suggesting that Ile-199 is gating the substrate cavity. Ile-199 is conserved in all known MAO B sequences except bovine MAO B, which has Phe in this position (the sequence of sheep MAO B is unknown). Phe is conserved in the analogous position in MAO A sequences. The human MAO B I199F mutant protein of MAO B binds to isatin (K(i) = 3 microM) but not to the three inhibitors listed above. The crystal structure of this mutant demonstrates that the side chain of Phe-199 interferes with the binding of those compounds. This suggests that the Ile-199 "gate" is a determinant for the specificity of these MAO B inhibitors and provides a molecular basis for the development of MAO B-specific reversible inhibitors without interference with MAO A function in neurotransmitter metabolism.

Animals↗
PubMed2004

Functional analysis of pyrimidine 5'-nucleotidase mutants causing nonspherocytic hemolytic anemia.

Inherited pyrimidine 5'-nucleotidase type I (P5'N-1) deficiency is the third most common erythrocyte enzymopathy that causes hemolysis. Fourteen different mutations have been identified to date. We have investigated the molecular bases of the disease by studying the biochemical properties of the recombinant wild-type human enzyme and 4 variant proteins (D87V, L131P, N179S, and G230R) bearing missense mutations found in patients affected by nonspherocytic hemolytic anemia. P5'N-1 is a relatively stable protein and has essentially identical catalytic efficiency toward cytidine monophosphate (CMP) and uridine monophosphate (UMP). All investigated mutant proteins display impaired catalytic properties and/or reduced thermostability, providing a rationale for the pathological effects of the mutations. Despite the substantial changes in the kinetic and thermostability parameters, the enzyme activity detected in the red blood cells of patients homozygous for mutations L131P and G230R exhibits moderate alterations. This suggests that P5'N-1 deficiency is compensated, possibly by other nucleotidases or alternative pathways in nucleotide metabolism. Therefore, nucleotidase activity may not be considered a prognostic indicator in patients affected by the enzymopathy.

5'-Nucleotidase↗
PubMed2004

Structures of Michaelis and product complexes of plant cytokinin dehydrogenase: implications for flavoenzyme catalysis.

Cytokinins form a diverse class of compounds that are essential for plant growth. Cytokinin dehydrogenase has a major role in the control of the levels of these plant hormones by catalysing their irreversible oxidation. The crystal structure of Zea mays cytokinin dehydrogenase displays the same two-domain topology of the flavoenzymes of the vanillyl-alcohol oxidase family but its active site cannot be related to that of any other family member. The X-ray analysis reveals a bipartite architecture of the catalytic centre, which consists of a funnel-shaped region on the protein surface and an internal cavity lined by the flavin ring. A pore with diameter of about 4A connects the two active-site regions. Snapshots of two critical steps along the reaction cycle were obtained through the structural analysis of the complexes with a slowly reacting substrate and the reaction product, which correspond to the states immediately before (Michaelis complex) and after (product complex) oxidation has taken place. The substrate displays a "plug-into-socket" binding mode that seals the catalytic site and precisely positions the carbon atom undergoing oxidation in close contact with the reactive locus of the flavin. A polarising H-bond between the substrate amine group and an Asp-Glu pair may facilitate oxidation. Substrate to product conversion results in small atomic movements, which lead to a planar conformation of the reaction product allowing double-bond conjugation. These features in the mechanism of amine recognition and oxidation differ from those observed in other flavin-dependent amine oxidases.

Adenine↗
PubMed2004

Crystal structure of a Baeyer-Villiger monooxygenase.

Flavin-containing Baeyer-Villiger monooxygenases employ NADPH and molecular oxygen to catalyze the insertion of an oxygen atom into a carbon-carbon bond of a carbonylic substrate. These enzymes can potentially be exploited in a variety of biocatalytic applications given the wide use of Baeyer-Villiger reactions in synthetic organic chemistry. The catalytic activity of these enzymes involves the formation of two crucial intermediates: a flavin peroxide generated by the reaction of the reduced flavin with molecular oxygen and the "Criegee" intermediate resulting from the attack of the flavin peroxide onto the substrate that is being oxygenated. The crystal structure of phenylacetone monooxygenase, a Baeyer-Villiger monooxygenase from the thermophilic bacterium Thermobifida fusca, exhibits a two-domain architecture resembling that of the disulfide oxidoreductases. The active site is located in a cleft at the domain interface. An arginine residue lays above the flavin ring in a position suited to stabilize the negatively charged flavin-peroxide and Criegee intermediates. This amino acid residue is predicted to exist in two positions; the "IN" position found in the crystal structure and an "OUT" position that allows NADPH to approach the flavin to reduce the cofactor. Domain rotations are proposed to bring about the conformational changes involved in catalysis. The structural studies highlight the functional complexity of this class of flavoenzymes, which coordinate the binding of three substrates (molecular oxygen, NADPH, and phenylacetone) in proximity of the flavin cofactor with formation of two distinct catalytic intermediates.

Acetone↗
PubMed2004

Crystal structure of human monoamine oxidase B, a drug target enzyme monotopically inserted into the mitochondrial outer membrane.

Monoamine oxidase B (MAO B) is an outer mitochondrial membrane protein that oxidizes arylalkylamine neurotransmitters and has been a valuable drug target for many neurological disorders. The 1.7 angstrom resolution structure of human MAO B shows the enzyme is dimeric with a C-terminal transmembrane helix protruding from each monomer and anchoring the protein to the membrane. This helix departs perpendicularly from the base of the structure in a different way with respect to other monotopic membrane proteins. Several apolar loops exposed on the protein surface are located in proximity of the C-terminal helix, providing additional membrane-binding interactions. One of these loops (residues 99-112) also functions in opening and closing the MAO B active site cavity, which suggests that the membrane may have a role in controlling substrate binding.

Crystallography, X-Ray↗