Membrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson’s disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs’ design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at −80 °C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.

Metal-dependent formate dehydrogenases reduce CO2 with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond. When this bond is closed, the enzyme is in an oxygen-tolerant resting state presenting almost no catalytic activity and very low formate affinity. Opening this bond triggers large conformational changes that propagate to the active site, resulting in high activity and high formate affinity, but also higher oxygen sensitivity. We present the structure of activated FdhAB and show that activity loss is associated with partial loss of the metal sulfido ligand. The redox switch mechanism is reversible in vivo and prevents enzyme reduction by physiological formate levels, conferring a fitness advantage during O2 exposure.

Desulfovibrio gigas aldehyde oxidoreductase (DgAOR) is a mononuclear molybdenum-containing enzyme from the xanthine oxidase (XO) family, a group of enzymes capable of catalyzing the oxidative hydroxylation of aldehydes and heterocyclic compounds. The kinetic studies reported in this work showed that DgAOR catalyzes the oxidative hydroxylation of aromatic aldehydes, but not heterocyclic compounds. NMR spectroscopy studies using C-13-labeled benzaldehyde confirmed that DgAOR catalyzes the conversion of aldehydes to the respective carboxylic acids. Steady-state kinetics in solution showed that high concentrations of the aromatic aldehydes produce substrate inhibition and in the case of 3-phenyl propionaldehyde a suicide substrate behavior. Hydroxyl-substituted aromatic aldehydes present none of these behaviors but the kinetic parameters are largely affected by the position of the OH group. High-resolution crystallographic structures obtained from single crystals of active-DgAOR soaked with benzaldehyde showed that the side chains of Phe(425) and Tyr(535) are important for the stabilization of the substrate in the active site. On the other hand, the X-ray data of DgAOR soaked with trans-cinnamaldehyde showed a cinnamic acid molecule in the substrate channel. The X-ray data of DgAOR soaked with 3-phenyl propionaldehyde showed clearly how high substrate concentrations inactivate the enzyme by binding covalently at the surface of the enzyme and blocking the substrate channel. The different reactivity of DgAOR versus aldehyde oxidase and XO towards aromatic aldehydes and N-heterocyclic compounds is explained on the basis of the present kinetic and structural data.

Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with density-functional theory calculations providing a unique view of the molybdenum active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of As(III) to the less toxic As(V) species.Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with density-functional theory calculations providing a unique view of the molybdenum active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of As(III) to the less toxic As(V) species.

Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens’ cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.

Abstract Vanadium compounds have frequently been proposed as therapeutics, but their application has been hampered by the lack of information on the different V-containing species that may form and how these interact with blood and cell proteins, and with enzymes. Herein, we report several resolved crystal structures of lysozyme with bound VIVO2+ and VIVOL2+, where L=2,2?-bipyridine or 1,10-phenanthroline (phen), and of trypsin with VIVO(picolinato)2 and VVO2(phen)+ moieties. Computational studies complete the refinement and shed light on the relevant role of hydrophobic interactions, hydrogen bonds, and microsolvation in stabilizating the structure. Noteworthy is that the trypsin?VVO2(phen) and trypsin?VIVO(OH)(phen) adducts correspond to similar energies, thus suggesting a possible interconversion under physiological/biological conditions. The obtained data support the relevance of hydrolysis of VIV and VV complexes in the several types of binding established with proteins and the formation of different adducts that might contribute to their pharmacological action, and significantly widen our knowledge of vanadium?protein interactions.

The mechanism of oxidation of N-heterocycle phthalazine to phthalazin-1(2H)-one and its associated free energy profile, catalyzed by human aldehyde oxidase (hAOX1), was studied in atomistic detail using QM/MM methodologies. The studied reaction was found to involve three sequential steps: (i) protonation of the substrate’s N2 atom by Lys893, (ii) nucleophilic attack of the hydroxyl group of the molybdenum cofactor (Moco) to the substrate, and (iii) hydride transfer from the substrate to the sulfur atom of the Moco. The free energy profile that was calculated revealed that the rate-limiting step corresponds to hydride transfer. It was also found that Lys893 plays a relevant role in the reaction, being important not only for the anchorage of the substrate close to the Moco, but also in the catalytic reaction. The variations of the oxidation state of the molybdenum ion throughout the catalytic cycle were examined too. We found out that during the displacement of the products away from the Moco, the transfer of electrons from the catalytic site to the FAD site was proton-coupled. As a consequence, the most favorable and fastest pathway for the enzyme to complete its catalytic cycle was that with MoV and a deprotonated SH ligand of the Moco with the FAD molecule converted to its semiquinone form, FADH•.The mechanism of oxidation of N-heterocycle phthalazine to phthalazin-1(2H)-one and its associated free energy profile, catalyzed by human aldehyde oxidase (hAOX1), was studied in atomistic detail using QM/MM methodologies. The studied reaction was found to involve three sequential steps: (i) protonation of the substrate’s N2 atom by Lys893, (ii) nucleophilic attack of the hydroxyl group of the molybdenum cofactor (Moco) to the substrate, and (iii) hydride transfer from the substrate to the sulfur atom of the Moco. The free energy profile that was calculated revealed that the rate-limiting step corresponds to hydride transfer. It was also found that Lys893 plays a relevant role in the reaction, being important not only for the anchorage of the substrate close to the Moco, but also in the catalytic reaction. The variations of the oxidation state of the molybdenum ion throughout the catalytic cycle were examined too. We found out that during the displacement of the products away from the Moco, the transfer of electrons from the catalytic site to the FAD site was proton-coupled. As a consequence, the most favorable and fastest pathway for the enzyme to complete its catalytic cycle was that with MoV and a deprotonated SH ligand of the Moco with the FAD molecule converted to its semiquinone form, FADH•.

Carbohydrate-binding-modules (CBMs) are discrete auxiliary protein modules with a non-catalytic carbohydrate-binding function and that exhibit a great diversity of binding specificities. CBMcarb-DB is a curated database that classifies the three-dimensional structures of CBM–carbohydrate complexes determined by single-crystal X-ray diffraction methods and solution NMR spectroscopy. We designed the database architecture and the navigation tools to query the database with the Protein Data Bank (PDB), UniProtKB, and GlyTouCan (universal glycan repository) identifiers. Special attention was devoted to describing the bound glycans using simple graphical representation and numerical format for cross-referencing to other glycosciences and functional data databases. CBMcarb-DB provides detailed information on CBMs and their bound oligosaccharides and features their interactions using several open-access applications. We also describe how the curated information provided by CBMcarb-DB can be integrated with AI algorithms of 3D structure prediction, facilitating structure–function studies. Also in this chapter, we discuss the exciting convergence of CBMcarb-DB with the glycan array repository, which serves as a valuable resource for investigating the specific binding interactions between glycans and various biomolecular targets. The interaction of the two fields represents a significant milestone in glycosciences. CBMcarb-DB is freely available at https://cbmdb.glycopedia.eu/ and https://cbmcarb.webhost.fct.unl.pt.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes ocular and urogenital infections in humans. The ability of C. trachomatis to grow intracellularly in a pathogen-containing vacuole (known as an inclusion) depends on chlamydial effector proteins transported into the host cell by a type III secretion system. Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes ocular and urogenital infections in humans. The ability of C. trachomatis to grow intracellularly in a pathogen-containing vacuole (known as an inclusion) depends on chlamydial effector proteins transported into the host cell by a type III secretion system. Among these effectors, several inclusion membrane proteins (Incs) insert in the vacuolar membrane. Here, we show that human cell lines infected by a C. trachomatis strain deficient for Inc CT288/CTL0540 (renamed IncM) displayed less multinucleation than when infected by IncM-producing strains (wild type or complemented). This indicated that IncM is involved in the ability of Chlamydia to inhibit host cell cytokinesis. The capacity of IncM to induce multinucleation in infected cells was shown to be conserved among its chlamydial homologues and appeared to require its two larger regions predicted to be exposed to the host cell cytosol. C. trachomatis-infected cells also displayed IncM-dependent defects in centrosome positioning, Golgi distribution around the inclusion, and morphology and stability of the inclusion. The altered morphology of inclusions containing IncM-deficient C. trachomatis was further affected by depolymerization of host cell microtubules. This was not observed after depolymerization of microfilaments, and inclusions containing wild-type C. trachomatis did not alter their morphology upon depolymerization of microtubules. Overall, these findings suggest that IncM may exert its effector function by acting directly or indirectly on host cell microtubules.

Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4′-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines.

A few ruthenium based metal carbonyl complexes, e.g. CORM-2 and CORM-3, have therapeutic activity attributed to their ability to deliver CO to biological targets. In this work, a series of related complexes with the formula [Ru(CO)(3)Cl2L] (L = DMSO (3), L-H3CSO(CH2)(2)CH(NH2)CO2H) (6a); D,L-H3CSO(CH2)(2)CH-(NH2)CO2H (6b); 3-NC5H4(CH2)(2)SO3.Na (7); 4-NC5H4(CH2)(2)SO3Na (8); PTA (9); DAPTA (10); H3CS-(CH2)(2)CH(OH) CO2H (11); CNCMe2CO2Me (12); CNCMeEtCO2Me (13); CN(c-C3H4)CO2Et) (14)) were designed, synthesized and studied. The effects of L on their stability, CO release profile, cytotoxicity and anti-inflammatory properties are described. The stability in aqueous solution depends on the nature of L as shown using HPLC and LC-MS studies. The isocyanide derivatives are the least stable complexes, and the S-bound methionine oxide derivative is the more stable one. The complexes do not release CO gas to the headspace, but release CO2 instead. X-ray diffraction of crystals of the model protein Hen Egg White Lysozyme soaked with 6b (4UWN) and 8 (4UWV) shows the addition of Ru-II(CO)(H2O)(4) at the His15 binding site. Soakings with 7 (4UWU) produced the metallacarboxylate [Ru(COOH)(CO)(H2O)(3)](+) bound to the His15 site. The aqueous chemistry of these complexes is governed by the water-gas shift reaction initiated with the nucleophilic attack of HO- on coordinated CO. DFT calculations show this addition to be essentially barrierless. The complexes have low cytotoxicity and low hemolytic indices. Following i.v. administration of CORM-3, the in vivo bio-distribution of CO differs from that obtained with CO inhalation or with heme oxygenase stimulation. A mechanism for CO transport and delivery from these complexes is proposed.