Human flavin-containing monooxygenase 3 (hFMO3) catalyses the oxygenation of a wide variety of compounds including drugs as well as dietary compounds. It is the major hepatic enzyme involved in the production of the N-oxide of trimethylamine (TMAO) and clinical studies have uncovered a striking correlation between plasma TMAO concentration and cardiovascular disease. Certain mutations within the hFMO3 gene cause defective trimethylamine (TMA) N-oxygenation leading to trimethylaminuria (TMAU) also known as fish-odour syndrome. In this paper, the inactivation mechanism of a TMAU-causing polymorphic variant, N61S, is investigated. Transient kinetic experiments show that this variant has a > 170-fold lower NADPH binding affinity than the wild type. Thermodynamic and spectroscopic experiments reveal that the poor NADP+ binding affinity accelerates the C4a-hydroperoxyFAD intermediate decay, responsible for an unfavourable oxygen transfer to the substrate. Steady-state kinetic experiments show significantly decreased N61S catalytic activity towards other substrates; methimazole, benzydamine and tamoxifen. The in vitro data are corroborated by in silico data where compared to the wild type enzyme, a hydrogen bond required for the stabilisation of the flavin intermediate is lacking. Taken together, the data presented reveal the molecular basis for the loss of function observed in N61S mutant.

Human hepatic flavin-containing monooxygenase 3 (hFMO3) catalyses the monooxygenation of carbon-bound reactive heteroatoms and plays an important role in the metabolism of drugs and xenobiotics. Although numerous hFMO3 allelic variants have been identified in patients and their biochemical properties well-characterised in vitro, the molecular mechanisms underlying loss-offunction mutations have still not been elucidated due to lack of detailed structural information of hFMO3. Therefore, in this work a 3D structural model of hFMO3 was generated by homology modeling, evaluated by a variety of different bioinformatics tools, refined by molecular dynamics simulations and further assessed based on in vitro biochemical data. The molecular dynamics simulation results highlighted 4 flexible regions of the protein with some of them overlapping the data from trypsin digest. This was followed by structural mapping of 12 critical polymorphic variants and molecular docking experiments with five different known substrates/drugs of hFMO3 namely, benzydamine, sulindac sulphide, tozasertib, methimazole and trimethylamine. Localisation of these mutations on the hFMO3 model provided a structural explanation for their observed biological effects and docked models of hFMO3-drug complexes gave insights into their binding mechanis m demonstrating that nitrogen-and sulfur-containing substrates interact with the isoalloxazine ring through Pi-Cation interaction and Pi-Sulfur interactions, respectively. Finally, the data presented give insights into the drug binding mechanism of hFMO3 which could be valuable not only for screening of new chemical entities but more significantly for designing of novel inhibitors of this important Phase I drug metabolising enzyme.

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A general review of spider burrows and history of their research in eighteenth to nineteenth centuries are provided on the basis of the literature, which is dispersed and almost forgotten by majority of ichnologists. Moreover, burrows of the wolf spider Trochosa hispanica Simon, 1870 from a mountain meadow in Albania are presented. They are composed of an almost straight through gently curved to slightly winding vertical shafts (8.2-17.2 mm in diameter) with a basal, oval chamber, which is 14.5-30.6 mm wide. Above the ground level, some of them show a low, agglutinated chimney a cone composed of soil granules. The burrows are 83-235 mm long. They are comparable with the trace fossil Macanopsis Macsotay, 1967. Other spider burrows can form a simple shaft, which may be ascribed to the ichnogenus Skolithos Haldeman, 1840, or a shaft with the side oblique branches, which is is similar to the ichnogenus Psilonichnus Fürsich, 1981. Many spider burrows show one or more chambers. Their outlet may be closed with a trapdoor or show a chimney sticking above the ground. They may show scratch traces running parallel to the burrow. The burrows are domiciles in which spiders spend a part of, or even the whole life. They protect spiders against harsh environmental conditions, foremost against too low or to high temperature, sheet floods, or predators. Moreover, they can be also a place for copulation, oviposition, parental care, placement of cocoons, or shedding the exuvia. Burrowing spider are more common in in warmer climatic zones, in open space, above the water ground level, and less common in flooded. So far, very few examples of fossil spider burrows are recognised, mostly in Cenozoic sediments, even if spiders are known since the Carboniferous.