TY - JOUR A1 - Mota, Cristiano A1 - Esmaeeli Moghaddam Tabalvandani, Mariam A1 - Coelho, Catarina A1 - Santos-Silva, Teresa A1 - Wolff, Martin A1 - Foti, Alessandro A1 - Leimkühler, Silke A1 - Romao, Maria Joao T1 - Human aldehyde oxidase (hAOX1) BT - structure determination of the Moco-free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms JF - FEBS Open Bio N2 - Human aldehyde oxidase (hAOX1) is a molybdenum enzyme with high toxicological importance, but its physiological role is still unknown. hAOX1 metabolizes different classes of xenobiotics and is one of the main drug-metabolizing enzymes in the liver, along with cytochrome P450. hAOX1 oxidizes and inactivates a large number of drug molecules and has been responsible for the failure of several phase I clinical trials. The interindividual variability of drug-metabolizing enzymes caused by single nucleotide polymorphisms (SNPs) is highly relevant in pharmaceutical treatments. In this study, we present the crystal structure of the inactive variant G1269R, revealing the first structure of a molybdenum cofactor (Moco)-free form of hAOX1. These data allowed to model, for the first time, the flexible Gate 1 that controls access to the active site. Furthermore, we inspected the thermostability of wild-type hAOX1 and hAOX1 with various SNPs (L438V, R1231H, G1269R or S1271L) by CD spectroscopy and ThermoFAD, revealing that amino acid exchanges close to the Moco site can impact protein stability up to 10 degrees C. These results correlated with biochemical and structural data and enhance our understanding of hAOX1 and the effect of SNPs in the gene encoding this enzyme in the human population. EnzymesAldehyde oxidase (); xanthine dehydrogenase (); xanthine oxidase (). DatabasesStructural data are available in the Protein Data Bank under the accession number . KW - human aldehyde oxidase KW - molybdenum cofactor KW - single nucleotide polymorphism KW - xanthine oxidase Y1 - 2019 U6 - https://doi.org/10.1002/2211-5463.12617 SN - 2211-5463 VL - 9 IS - 5 SP - 925 EP - 934 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Zupok, Arkadiusz A1 - Górka, Michał Jakub A1 - Siemiatkowska, Beata A1 - Skirycz, Aleksandra A1 - Leimkühler, Silke T1 - Iron-Dependent Regulation of Molybdenum Cofactor Biosynthesis Genes in Escherichia coli JF - Journal of bacteriology N2 - Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In recent years it has become obvious that the availability of iron plays an important role in the biosynthesis of Moco. First, the MoaA protein binds two (4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional NFe-4S) cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is a shared protein with a main role in the assembly of Fe-S clusters. In this report, we investigated the transcriptional regulation of the moaABCDE operon by focusing on its dependence on cellular iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, our data show that the regulation of the moaABCDE operon at the level of transcription is only marginally influenced by the availability of iron. Nevertheless, intracellular levels of Moco were decreased under iron-limiting conditions, likely based on an inactive MoaA protein in addition to lower levels of the L-cysteine desulfurase IscS, which simultaneously reduces the sulfur availability for Moco production. IMPORTANCE FNR is a very important transcriptional factor that represents the master switch for the expression of target genes in response to anaerobiosis. Among the FNR-regulated operons in Escherichia coli is the moaABCDE operon, involved in Moco biosynthesis. Molybdoenzymes have essential roles in eukaryotic and prokaryotic organisms. In bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. This work investigates the connection of iron availability to the biosynthesis of Moco and the production of active molybdoenzymes. KW - Escherichia coli KW - FNR KW - iron regulation KW - iron-sulfur cluster KW - anaerobic respiration KW - molybdenum cofactor Y1 - 2019 U6 - https://doi.org/10.1128/JB.00382-19 SN - 0021-9193 SN - 1098-5530 VL - 201 IS - 17 PB - American Society for Microbiology CY - Washington ER -