A pH-dependent shift of redox cofactor specificity in a benzyl alcohol dehydrogenase of aromatoleum aromaticum EbN1.

We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD+ or NADP+ as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2'-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp3-hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol. KEY POINTS: •Zn-containing BaDH has activity with either NAD + or NADP+ at different pH optima. •BaDH converts a broad range of substrates. •BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol.

A bacterial tungsten-containing aldehyde oxidoreductase forms an enzymatic decorated protein nanowire.

Aldehyde oxidoreductases (AORs) are tungsten enzymes catalyzing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium Aromatoleum aromaticum (AORAa) consists of three different subunits (AorABC) and uses nicotinamide adenine dinucleotide (NAD) as an electron acceptor. Here, we reveal that the enzyme forms filaments of repeating AorAB protomers that are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy. The polyferredoxin-like subunit AorA oligomerizes to an electron-conducting nanowire that is decorated with enzymatically active and W-cofactor (W-co) containing AorB subunits. Our structure further reveals the binding mode of the native substrate benzoate in the AorB active site. This, together with quantum mechanics:molecular mechanics (QM:MM)-based modeling for the coordination of the W-co, enables formulation of a hypothetical catalytic mechanism that paves the way to further engineering for applications in synthetic biology and biotechnology.

Electrocatalytic Aldehyde Oxidation by a Tungsten Dependent Aldehyde Oxidoreductase from Aromatoleum Aromaticum.

In contrast to their molybdenum dependent relatives, tungsten enzymes operate at significantly lower redox potentials, and in some cases they can carry out reversible redox transformations of their substrates and products. Still, the electrochemical properties of W enzymes have received much less attention than their Mo relatives. Herein we analyse the tungsten enzyme aldehyde oxidoreductase (AOR) from the mesophilic bacterium Aromatoleum aromaticum which has been immobilised on a glassy carbon working electrode. This generates a functional system that electrochemically oxidises a wide variety of aromatic and aliphatic aldehydes in the presence of the electron transfer mediators benzyl viologen, methylene blue or dichlorophenol indophenol. Simulation of the cyclic voltammetry has enabled a thorough kinetic analysis of the system, which reveals that methylene blue acts as a two-electron acceptor. In contrast, the other two mediators act as single electron oxidants. The different electrochemical driving forces imparted by these mediators also lead to significantly different outer sphere electron transfer rates with AOR. This work shows that electrocatalytic aldehyde oxidation can be achieved at a low applied electrochemical potential leading to an extremely energy efficient catalytic process.

Tungsten Enzyme Using Hydrogen as an Electron Donor to Reduce Carboxylic Acids and NAD.

Tungsten-dependent aldehyde oxidoreductases (AORs) catalyze the oxidation of aldehydes to acids and are the only known enzymes reducing non-activated acids using electron donors with low redox potentials. We report here that AOR from Aromatoleum aromaticum (AOR Aa ) catalyzes the reduction of organic acids not only with low-potential Eu(II) or Ti(III) complexes but also with H2 as an electron donor. Additionally, AOR Aa catalyzes the H2-dependent reduction of NAD+ or benzyl viologen. The rate of H2-dependent NAD+ reduction equals to 10% of that of aldehyde oxidation, representing the highest H2 turnover rate observed among the Mo/W enzymes. As AOR Aa simultaneously catalyzes the reduction of acids and NAD+, we designed a cascade reaction utilizing a NAD(P)H-dependent alcohol dehydrogenase to reduce organic acids to the corresponding alcohols with H2 as the only reductant. The newly discovered W-hydrogenase side activity of AOR Aa may find applications in either NADH recycling or conversion of carboxylic acids to more useful biochemicals.

Use of a FluoroType® System for the Rapid Detection of Patients with Multidrug-Resistant Tuberculosis-State of the Art Case Presentations.

According to the World Health Organization (WHO), there were 465,000 cases of tuberculosis caused by strains resistant to at least two first-line anti-tuberculosis drugs: rifampicin and isoniazid (MDR-TB). In light of the growing problem of drug resistance in Mycobacterium tuberculosis across laboratories worldwide, the rapid identification of drug-resistant strains of the Mycobacterium tuberculosis complex poses the greatest challenge. Progress in molecular biology and the development of nucleic acid amplification assays have paved the way for improvements to methods for the direct detection of Mycobacterium tuberculosis in specimens from patients. This paper presents two cases that illustrate the implementation of molecular tools in the recognition of drug-resistant tuberculosis.

Pulmonary actinomycosis complicated by fistula of the chest wall.

Actinomycosis is a rare disease caused by Actinomyces spp. The clinical and radiological picture of the disease is uncharacter-istic, which delays the diagnosis and can lead to complications. We present a case of pulmonary actinomycosis complicated by a chest wall fistula in a 43-year-old man with advanced tooth decay. The patient was admitted to our Department due to a chest wall fistula with bloody discharge. A few months earlier, he was treated with antibiotics for pneumonia. Since then, weakness, exertional dyspnoea, and weight loss had been observed. On admission, increased inflammatory markers were found in laboratory tests. Chest computed tomography (CT) revealed right-sided encapsulated pleural fluid collection containing gas bubbles, pleural thickening, anterior thoracic wall soft tissues thickening and subcutaneous fat stranding. CT suggested an empyema or a breast either pleural malignancy. The picture suggested a breast or pleural tumour to differentiate with an empyema. Videothoracoscopy was performed, the histological examination of the collected samples revealed granulation tissue and bacterial colony of a morphology corresponding to Actinomyces spp. Pulmonary actinomycosis was diagnosed. Antibiotic therapy according to the guidelines was initiated and dental treatment was recommended. Healing of the fistula and significant regression of lesions in the right lung were achieved. Although it is a rare disease, actinomycosis should be considered in the differential diagnosis of any chronic infiltrative lung lesions.

Investigation of quaternary structure of aggregating 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans: In the pursuit of consensus of various biophysical techniques.

In this work we analyzed the quaternary structure of FAD-dependent 3-ketosteroid dehydrogenase (AcmB) from Sterolibacterium denitrificans, the protein that in solution forms massive aggregates (>600 kDa). Using size-excursion chromatography (SEC), dynamic light scattering (DLS), native-PAGE and atomic force microscopy (AFM) we studied the nature of enzyme aggregation. Partial protein de-aggregation was facilitated by the presence of non-ionic detergent such as Tween 20 or by a high degree of protein dilution but not by addition of a reducing agent or an increase of ionic strength. De-aggregating influence of Tween 20 had no impact on either enzyme's specific activity or FAD reconstitution to recombinant AcmB. The joint experimental (DLS, isoelectric focusing) and theoretical investigations demonstrated gradual shift of enzyme's isoelectric point upon aggregation from 8.6 for a monomeric form to even 5.0. The AFM imaging on mica or highly oriented pyrolytic graphite (HOPG) surface enabled observation of individual protein monomers deposited from a highly diluted solution (0.2 µg/ml). Such approach revealed that native AcmB can indeed be monomeric. AFM imaging supported by theoretical random sequential adsorption (RSA) kinetics allowed estimation of distribution enzyme forms in the bulk solution: 5%, monomer, 11.4% dimer and 12% trimer. Finally, based on results of AFM as well as analysis of the surface of AcmB homology models we have observed that aggregation is most probably initiated by hydrophobic forces and then assisted by electrostatic attraction between negatively charged aggregates and positively charged monomers.

Characterization of an Aldehyde Oxidoreductase From the Mesophilic Bacterium Aromatoleum aromaticum EbN1, a Member of a New Subfamily of Tungsten-Containing Enzymes.

The biochemical properties of a new tungsten-containing aldehyde oxidoreductase from the mesophilic betaproteobacterium Aromatoleum aromaticum EbN1 (AOR Aa ) are presented in this study. The enzyme was purified from phenylalanine-grown cells of an overexpressing mutant lacking the gene for an aldehyde dehydrogenase normally involved in anaerobic phenylalanine degradation. AOR Aa catalyzes the oxidation of a broad variety of aldehydes to the respective acids with either viologen dyes or NAD+ as electron acceptors. In contrast to previously known AORs, AOR Aa is a heterohexameric protein consisting of three different subunits, a large subunit containing the W-cofactor and an Fe-S cluster, a small subunit containing four Fe-S clusters, and a medium subunit containing an FAD cofactor. The presence of the expected cofactors have been confirmed by elemental analysis and spectrophotometric methods. AOR Aa has a pH optimum of 8.0, a temperature optimum of 40°C and is completely inactive at 50°C. Compared to archaeal AORs, AOR Aa is remarkably resistant against exposure to air, exhibiting a half-life time of 1 h as purified enzyme and being completely unaffected in cell extracts. Kinetic parameters of AOR Aa have been obtained for the oxidation of one aliphatic and two aromatic aldehydes, resulting in about twofold higher k cat values with benzyl viologen than with NAD+ as electron acceptor. Finally, we obtained evidence that AOR Aa is also catalyzing the reverse reaction, reduction of benzoate to benzaldehyde, albeit at very low rates and under conditions strongly favoring acid reduction, e.g., low pH and using Ti(III) citrate as electron donor of very low redox potential. AOR Aa appears to be a prototype of a new subfamily of bacterial AOR-like tungsten-enzymes, which differ from the previously known archaeal AORs mostly by their multi-subunit composition, their low sensitivity against oxygen, and the ability to use NAD+ as electron acceptor.