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Light-dependent or light-stimulated catalysis provides a multitude of perspectives for implementation in technological or biomedical applications. Despite substantial progress made in the field of photobiocatalysis, the number of usable light-responsive enzymes is still very limited. Flavoproteins have exceptional potential for photocatalytic applications because the name-giving cofactor intrinsically features light-dependent reactivity, undergoing photoreduction with a variety of organic electron donors. However, in the vast majority of these enzymes, photoreactivity of the enzyme-bound flavin is limited or even suppressed. Here, we present a flavoprotein monooxygenase in which catalytic activity is controllable by blue light illumination. The reaction depends on the presence of nicotinamide nucleotide-type electron donors, which do not support the reaction in the absence of light. Employing various experimental approaches, we demonstrate that catalysis depends on a protein-mediated photoreduction of the flavin cofactor, which proceeds via a radical mechanism and a transient semiquinone intermediate., The number of usable light-responsive enzymes is limited, despite the potential biotechnological applications. Here, the authors report a flavoprotein monooxygenase which is controllable by blue light illumination, and propose a mechanism involving protein-mediated radical photoreduction of FAD via a semiquinone intermediate.

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (Delta T-m >= +3 degrees C). By combining mutations, a 10-fold mutant was generated with a T-m of 94 degrees C (+51 degrees C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a T-m of 88 degrees C (+45 degrees C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.

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.

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. © 2016 Wiley Periodicals, Inc.

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.

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. © 2016 Wiley Periodicals, Inc.

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. 2016 Elsevier Masson SAS. All rights reserved. This work was supported by internal grants (# QUST-CPH-FALL-14?15-9 and QUST-CPH-SPR-14/15-9 ) from the Office of Academic Research, Qatar University , Doha, Qatar. C.B. is member of the collaborative network within the COST Action CM1103, Structure-based drug design for diagnosis and treatment of neurological diseases . We are grateful to Professor Neal Castagnoli Jr. Professor Emeritus, Virginia Tech for providing the MMTP substrate. The 2D-NMR spectra were recorded by Professor Khalid Elsayed, University of Louisiana at Monroe, USA. Scopus

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 A(3) volume and MAO B contains a dipartite cavity structure with volumes of ~290 A(3) (entrance cavity) and ~400 A(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 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 K(M) but unaltered k(cat) values. The altered K(M) 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.

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 (similar to 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 8 alpha-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.

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 Angstrom), 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 Angstrom). 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.

Alditol oxidase (AldO) from Streptomyces coelicolor A3(2) is a soluble monomeric flavin-dependent oxidase that performs selective oxidation of the terminal primary hydroxyl group of several alditols. Here, we report the crystal structure of the recombinant enzyme in its native state and in complex with both six-carbon (mannitol and sorbitol) and five-carbon substrates (xylitol). AldO shares the same folding topology of the members of the vanillyl-alcohol oxidase family of flavoenzymes and exhibits a covalently linked FAD which is located at the bottom of a funnel-shaped pocket that forms the active site. The high resolution of the three-dimensional structures highlights a well-defined hydrogen-bonding network that tightly constrains the substrate in the productive conformation for catalysis. Substrate binding occurs through a lock-and-key mechanism and does not induce conformational changes with respect to the ligand-free protein. A network of charged residues is proposed to favor catalysis through stabilization of the deprotonated form of the substrate. A His side chain acts as back door that "pushes" the substrate-reactive carbon atom toward the N5-C4a locus of the flavin. Analysis of the three-dimensional structure reveals possible pathways for diffusion of molecular oxygen and a small cavity on the re side of the flavin that may host oxygen during FAD reoxidation. These features combined with the tight shape of the catalytic site provide insights into the mechanism of AldO-mediated regioselective oxidation reactions and its substrate specificity.

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 angstrom resolution. The crystals belong to space group C2, with unit-cell parameters a = 107, b = 68, c = 58 angstrom, 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 angstrom.

The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (k(cat)/K-m) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phoshoryl-group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP forming 3-phosphoglycerate (3-PG) and ATP. PGK is a typical two-domain hinge-bending enzyme, with a highly conserved structure. The N-terminal domain binds 1,3-BPG/3-PG, whereas the C-terminal domain binds Mg-ADP/Mg-ATP.Humans have two PGK isozymes, PGK1 and PGK2, where PGK1 is an ubiquitous enzyme that is expressed in all somatic cells and PGK2 is a testis-specific enzyme. The PGK1 gene is located on the X-chromosome q-13.1, contains 11 exons and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in an enzyme deficiency that is for the most clinically characterized by mild-to severe hemolytic anemia and various defects in the central nervous system. To date, 19 different mutations with worldwide distribution have been reported. No correlation between the residual PGK activity and the severity of the clinical manifestations have been documented so far. To analyze the mutations at protein level and possibly to correlate the genotype to clinical phenotype, we started with the molecular characterization of the wild-type PGK1 enzyme and three mutants (I47N, D164 and S320N) obtained from E.coli as recombinant proteins. The corresponding mutations, i.e., c.140T>A, c.491A>T and c.959G>A, have been identified in patients with PGK deficiency and affected by severe hemolytic anemia and progressive mental retardation. The cDNA encoding the PGK1 was prepared starting from a blood sample of a healthy donor, with normal PGK1 activity. Site-directed mutagenesis was used to introduce the desired mutations into the PGK1 cDNA. The wild type enzyme was expressed to its maximum level (about 80–100 mg of enzyme per liter of culture) after 5 hours of induction with 0.5 mM IPTG at 37 °C. For mutant enzymes the induction temperature was lowered to 25°C. All recombinant enzymes were purified to homogeneity after a single chromatographic step on DEAE Sepharose column. The wild-type enzyme was crystallized in both free form or complexed with 3-PG. The corresponding structures were solved to high resolution (1.8 and 1.6 A, respectively) and compared. Essentially, binding 3-PG caused a 6° rotation of the N-domain in respect to the C-domain. The recombinant enzyme exhibited kinetic properties similar to those of the authentic enzyme, displaying vs 3-PG and ATP alike specific activities (about 1000 U/mg) and alike Km values (about 1mM). I47N and S320N mutant enzymes showed kcat values 3-fold lower than the wild-type enzyme. The D164V was characterized by a Km value vs 3-PG 15 times higher than that of the other enzymes studied and a catalytic efficiency 70 times lower. Finally, all mutant enzymes turned out to be highly heat unstable with respect to the wildtype enzyme, losing half of their activity after approximately 10 minutes of incubation at 37 °C. At higher temperatures, the wild-type enzyme was protected from heat inactivation by Mg-ATP or 3-PG. On the contrary, no one mutant was protect by Mg-ATP and the D164V and S320N mutants were not even protected by 3-PG. Therefore, these preliminary studies indicate that all mutations target amino acid residues located in positions primarily important for preserving the protein stability during the red cell life span.