Stefano Rovida

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 Å), 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 Å). PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded β-sheet and a capping α-helix. The tightly bound FAD prosthetic group (Kd = 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.

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 Å3 volume and MAO B contains a dipartite cavity structure with volumes of ∼ 290 Å3 (entrance cavity) and ∼ 400 Å3 (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 data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared with wild‐type enzyme while the Ile199Ala‐Tyr326Ala MAO B mutant exhibits alterations in residues 100–103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine KM but unaltered kcat values. The altered KM values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to wild‐type enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala‐Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate that the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provide insights into specific reversible inhibitor design for these membrane‐bound enzymes.

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. All compounds appeared to be selective MAO-B inhibitors with Ki values in the micromolar to submicromolar range. Molecular modeling studies have been performed to get insight into the binding mode of the synthesized compounds to human MAO-B active site.

Polycyclic Ketone Monooxygenase from the Thermophilic Fungus Thermothelomyces thermophila: A Structurally Distinct Biocatalyst for Bulky Substrates

Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a well-established tool to address these challenges, but known BVMOs have shortcomings in either stability or substrate selectivity. We characterized a novel BVMO from the thermophilic fungus Thermothelomyces thermophila, determined its three-dimensional structure, and demonstrated its use as a promising biocatalyst. This fungal enzyme displays excellent enantioselectivity, acts on various ketones, and is particularly active on polycyclic molecules. Most notably we observed that the enzyme can perform oxidations on both the A and D ring when converting steroids. These functional properties can be linked to unique structural features, which identify enzymes acting on bulky substrates as a distinct subgroup of the BVMO class.

Structure-Based Redesign of Cofactor Binding in Putrescine Oxidase

Putrescine oxidase (PuO) from Rhodococcus erythropolis is a soluble homodimeric flavoprotein, which oxidizes small aliphatic diamines. In this study, we report the crystal structures and cofactor binding properties of wild-type and mutant enzymes. From a structural viewpoint, PuO closely resembles the sequence-related human monoamine oxidases A and B. This similarity is striking in the flavin-binding site even if PuO does not covalently bind the cofactor as do the monoamine oxidases. A remarkable conserved feature is the cis peptide conformation of the Tyr residue whose conformation is important for substrate recognition in the active site cavity. The structure of PuO in complex with the reaction product reveals that Glu324 is crucial in recognizing the terminal amino group of the diamine substrate and explains the narrow substrate specificity of the enzyme. The structural analysis also provides clues for identification of residues that are responsible for the competitive binding of ADP versus FAD (~50% of wild-type PuO monomers isolated are occupied by ADP instead of FAD). By replacing Pro15, which is part of the dinucleotide-binding domain, enzyme preparations were obtained that are almost 100% in the FAD-bound form. Furthermore, mutants have been designed and prepared that form a covalent 8α-S-cysteinyl-FAD linkage. These data provide new insights into the molecular basis for substrate recognition in amine oxidases and demonstrate that engineering of flavoenzymes to introduce covalent linkage with the cofactor is a possible route to develop more stable protein molecules, better suited for biocatalytic purposes.

Pyrrole- and indole-containing tranylcypromine derivatives as novel lysine-specific demethylase 1 inhibitors active on cancer cells

On the basis of previous research showing the capability of N-carbobenzyloxy-(Z-)amino acid-tranylcypromine (-TCPA) derivatives to inhibit LSD1, we inserted at the 4-amino-TCPA moiety first a Z-Pro (9) and a Z-Gly (10) residue and then, after the encouraging data obtained for 9, a pyrrole and an indole ring in which the relative N1 position carried a acetophenone, a N-phenyl/benzylacetamide, or a Z chain (11a–f and 12a–f, respectively). In both series, the Z-pyrrole and indole derivatives 11e, f and 12e, f displayed high LSD1 inhibitory activity. The compounds are able to inhibit LSD1 in NB4 cells, increasing the expression of two related genes, GFI-1b and ITGAM, and to induce cell growth arrest in the AML MB4-11 and APL NB4 cell lines.

Structural and biochemical insights into 7β-hydroxysteroid dehydrogenase stereoselectivity.

Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo‐ and regio‐selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7β‐hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active‐site accessibility, the bases of the specificity for NADP+, and the general architecture of the steroid binding site. Comparison with 7α‐hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C‐terminal extension reshapes the substrate site of the β‐selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859–865.

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.