Systematic exploration of predicted destabilizing nonsynonymous single nucleotide polymorphisms (nsSNPs) of human aldehyde oxidase: A Bio-informatics study.

Aldehyde Oxidase (hAOX1) is a cytosolic enzyme involved in the metabolism of drugs and xenobiotic compounds. The enzyme belongs to the xanthine oxidase (XO) family of Mo containing enzyme and is a homo-dimer of two 150 kDa monomers. Nonsynonymous Single Nucleotide Polymorphisms (nsSNPs) of hAOX1 have been reported as affecting the ability of the enzyme to metabolize different substrates. Some of these nsSNPs have been biochemically and structurally characterized but the lack of a systematic and comprehensive study regarding all described and validated nsSNPs is urgent, due to the increasing importance of the enzyme in drug development, personalized medicine and therapy, as well as in pharmacogenetic studies. The objective of the present work was to collect all described nsSNPs of hAOX1 and utilize a series of bioinformatics tools to predict their effect on protein structure stability with putative implications on phenotypic functional consequences. Of 526 nsSNPs reported in NCBI-dbSNP, 119 are identified as deleterious whereas 92 are identified as nondeleterious variants. The stability analysis was performed for 119 deleterious variants and the results suggest that 104 nsSNPs may be responsible for destabilizing the protein structure, whereas five variants may increase the protein stability. Four nsSNPs do not have any impact on protein structure (neutral nsSNPs) of hAOX1. The prediction results of the remaining six nsSNPs are nonconclusive. The in silico results were compared with available experimental data. This methodology can also be used to identify and prioritize the stabilizing and destabilizing variants in other enzymes involved in drug metabolism.

The crystal structure of Cupriavidus necator nitrate reductase in oxidized and partially reduced states.

The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe-4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C(4)mim]Cl(-). In addition, UV-Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.