Identification of fungal dihydrouracil-oxidase genes by expression in Saccharomyces cerevisiae

Bouwknegt, Jonna; Vos, Aurin M.; Ortiz‐Merino, Raúl A.; Cuylenburg, Daphne C. van; Luttik, Marijke A. H.; Pronk, Jack T.

doi:10.1007/s10482-022-01779-9

Cited by 1 publication

(11 citation statements)

References 79 publications

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“…Intriguingly, all dematiaceous enzymes complemented ura1Δ growth using DHU, implicating them as DHUO, but pyrimidine prototrophy was not identical for each DHUO at equivalent copper levels, suggestive of variation in oxidase activity between enzymes of different species. In line with previous findings [ 53 ], none of the dematiaceous mould enzymes could use DHU to rescue pyrimidine auxotrophy in anaerobic conditions ( Fig 7B ). These data show that these putative enzymes use DHU and oxygen to produce uracil, implicating them as DHUO rather than DHODH.…”

Section: Resultssupporting

confidence: 93%

“…The previously characterised A . alternata DHUO [ 53 ] rescued the auxotrophy well at 250 μM copper and above. Intriguingly, all dematiaceous enzymes complemented ura1Δ growth using DHU, implicating them as DHUO, but pyrimidine prototrophy was not identical for each DHUO at equivalent copper levels, suggestive of variation in oxidase activity between enzymes of different species.…”

Section: Resultsmentioning

confidence: 99%

“…While our work was underway, a study used yeast complementation and lysate assays to demonstrate that the Ura1-like gene from A . alternata encodes a DHUO: a redox enzyme that utilises oxygen to convert DHU or dihydrothymine into uracil or thymine, plus hydrogen peroxide [ 53 ]. In line with this work, we subsequently found that the putative dematiaceous enzymes facilitated pyrimidine prototrophy in yeast when supplemented with DHU but only under aerobic conditions.…”

Section: Discussionmentioning

confidence: 99%

“…Our observations corroborate this; both dematiaceous enzymes bind FMN and cannot rescue yeast growth in anaerobic conditions even in the presence of DHU. Expanding on preliminary sequence analysis [ 53 ], all class 1A DHODH and DHUO proteins assessed in this study shared a highly conserved active site that is likely a large contribution to the in silico assignment of the latter as the former in protein databases. Further, we provide the first insight into the mechanism of DHU oxidation through the identification of a catalytic cysteine in the A .…”

Section: Discussionmentioning

confidence: 99%

“…Work published during the course of this study revealed a class 1A DHODH-like gene in A . alternata to be a dihydrouracil oxidase (DHUO) [ 53 ], a poorly characterised type of enzyme described in only one other genus of fungi [ 54 ]. DHUO have been seen to convert dihydrouracil (DHU) and dihydrothymine to uracil and thymine, respectively, resembling a step that is the reverse of the initial reaction in the reductive pathway of pyrimidine degradation.…”

Section: Introductionmentioning

confidence: 99%

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Characterisation of putative class 1A DHODH-like proteins from Mucorales and dematiaceous mould species

Pinder¹,

Lebedinec²,

Levine³

et al. 2023

PLoS ONE

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Olorofim is a new antifungal in clinical development which has a novel mechanism of action against dihydroorotate dehydrogenase (DHODH). DHODH form a ubiquitous family of enzymes in the de novo pyrimidine biosynthetic pathway and are split into class 1A, class 1B and class 2. Olorofim specifically targets the fungal class 2 DHODH present in a range of pathogenic moulds. The nature and number of DHODH present in many fungal species have not been addressed for large clades of this kingdom. Mucorales species do not respond to olorofim; previous work suggests they have only class 1A DHODH and so lack the class 2 target that olorofim inhibits. The dematiaceous moulds have mixed susceptibility to olorofim, yet previous analyses imply that they have class 2 DHODH. As this is at odds with their intermediate susceptibility to olorofim, we hypothesised that these pathogens may maintain a second class of DHODH, facilitating pyrimidine biosynthesis in the presence of olorofim. The aim of this study was to investigate the DHODH repertoire of clinically relevant species of Mucorales and dematiaceous moulds to further characterise these pathogens and understand variations in olorofim susceptibility. Using bioinformatic analysis, S. cerevisiae complementation and biochemical assays of recombinant protein, we provide the first evidence that two representative members of the Mucorales have only class 1A DHODH, substantiating a lack of olorofim susceptibility. In contrast, bioinformatic analyses initially suggested that seven dematiaceous species appeared to harbour both class 1A-like and class 2-like DHODH genes. However, further experimental investigation of the putative class 1A-like genes through yeast complementation and biochemical assays characterised them as dihydrouracil oxidases rather than DHODHs. These data demonstrate variation in dematiaceous mould olorofim susceptibility is not due to a secondary DHODH and builds on the growing picture of fungal dihydrouracil oxidases as an example of horizontal gene transfer.

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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