Investigating the amino acid sequences of membrane bound dihydroorotate:quinone oxidoreductases (DHOQOs): Structural and functional implications

Sousa, Filipe de; Refojo, Patrícia N.; Pereira, Manuela M.

doi:10.1016/j.bbabio.2020.148321

Cited by 13 publications

(5 citation statements)

References 57 publications

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“…It is, however, interesting to note that the distribution of the cationic residues in the α1-α2 microdomain does not seem highly conserved in the Class II DHODHs in Figure 10 , spanning a wide evolutionary distance. This conclusion is in line with a recent comprehensive bioinformatics study by Sousa et al [ 76 ]. None of the positively charged residues on the outer surface of the α1-α2 microdomain were pointed out as highly conserved in an alignment of 1062 Class II DHODH sequences, or the whole α1-α2 microdomain, was one of the least conserved regions of the enzymes.…”

Section: Discussionsupporting

confidence: 92%

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition

Rodriguez

Wacklin-Knecht

Clifton

et al. 2022

IJMS

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The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH (HsΔ29DHODH) and the DHODH from Escherichia coli (EcDHODH) with ubiquinone (Q10) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q10. Q10 is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q10. We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q10 is mediated by enzyme penetration. We can also show that EcDHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than HsΔ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q10, as well as lipid composition, on enzyme binding.

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Section: Discussionsupporting

confidence: 92%

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition

Rodriguez

Wacklin-Knecht

Clifton

et al. 2022

IJMS

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“…A cysteine as catalytic base is considered a hallmark of Class-I DHODs [ 57 ] while, with few reported exceptions, Class-II enzymes have a serine in this position [ 16 ]. Instead, the three anaerobically active Ura9 orthologs investigated in this study had a Cys in the catalytic-base position and shared this feature with only 3 of 331 other predicted fungal Ura9 orthologs.…”

Section: Discussionmentioning

confidence: 99%

“…Based on analysis of 1500 Class II DHOD sequences and structural alignments, Sousa et al . [ 16 ] proposed conserved residues involved in quinone binding, stabilizing and pocket entry (Additional file 5 : Figure S2). Ura9 sequences from Neocallimastigomycetes lacked several of these conserved residues as a consequence of their N-terminal truncation.…”

Section: Discussionmentioning

confidence: 99%

“…Most eukaryotes harbor a monomeric Class-II DHOD that donates electrons to the quinone pool of the mitochondrial respiratory chain [ 8 , 14 – 16 ]. Bacterial Class-II DHODs have an N-terminal sequence that localizes them to the inside of the cytoplasmic membrane, whereas eukaryotic enzymes are targeted to the outside of the mitochondrial inner membrane [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

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Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

Bouwknegt

Koster

Vos

et al. 2021

Fungal Biol Biotechnol

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Background In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD. Results Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9. Conclusions Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure–function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.

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“…Dihydroorotate dehydrogenases are involved in pyrimidine biosynthesis but are also involved in energy metabolism. These flavin mononucleotide-containing enzymes facilitate the oxidation of dihydroorotate to orotate and transfer those electrons to quinones (Sousa et al, 2021). AKL02_07710 is annotated as a DUF59 domain-containing protein and BLASTP indicated this protein in ElOx9 T has nearly 100% homology to SUF system Fe-S cluster assembly proteins found in a variety of Thioclava species.…”

Section: Essential Genes For Extracellular Electron Transfermentioning

confidence: 99%

Genome-Scale Mutational Analysis of Cathode-Oxidizing Thioclava electrotropha ElOx9T

et al. 2022

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Extracellular electron transfer (EET) – the process by which microorganisms transfer electrons across their membrane(s) to/from solid-phase materials – has implications for a wide range of biogeochemically important processes in marine environments. Though EET is thought to play an important role in the oxidation of inorganic minerals by lithotrophic organisms, the mechanisms involved in the oxidation of solid particles are poorly understood. To explore the genetic basis of oxidative EET, we utilized genomic analyses and transposon insertion mutagenesis screens (Tn-seq) in the metabolically flexible, lithotrophic Alphaproteobacterium Thioclava electrotropha ElOx9T. The finished genome of this strain is 4.3 MB, and consists of 4,139 predicted ORFs, 54 contain heme binding motifs, and 33 of those 54 are predicted to localize to the cell envelope or have unknown localizations. To begin to understand the genetic basis of oxidative EET in ElOx9T, we constructed a transposon mutant library in semi-rich media which was comprised of >91,000 individual mutants encompassing >69,000 unique TA dinucleotide insertion sites. The library was subjected to heterotrophic growth on minimal media with acetate and autotrophic oxidative EET conditions on indium tin oxide coated glass electrodes poised at –278 mV vs. SHE or un-poised in an open circuit condition. We identified 528 genes classified as essential under these growth conditions. With respect to electrochemical conditions, 25 genes were essential under oxidative EET conditions, and 29 genes were essential in both the open circuit control and oxidative EET conditions. Though many of the genes identified under electrochemical conditions are predicted to be localized in the cytoplasm and lack heme binding motifs and/or homology to known EET proteins, we identified several hypothetical proteins and poorly characterized oxidoreductases that implicate a novel mechanism(s) for EET that warrants further study. Our results provide a starting point to explore the genetic basis of novel oxidative EET in this marine sediment microbe.

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Investigating the amino acid sequences of membrane bound dihydroorotate:quinone oxidoreductases (DHOQOs): Structural and functional implications

Cited by 13 publications

References 57 publications

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition

Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

Genome-Scale Mutational Analysis of Cathode-Oxidizing Thioclava electrotropha ElOx9T

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