Mitochondrial Carriers Link the Catabolism of Hydroxyaromatic Compounds to the Central Metabolism in Candida parapsilosis

The pathogenic yeast Candida parapsilosis metabolizes hydroxyderivatives of benzene and benzoic acid to compounds channeled into central metabolism, including the mitochondrially localized tricarboxylic acid cycle, via the 3-oxoadipate and gentisate pathways. The orchestration of both catabolic pathways with mitochondrial metabolism as well as their evolutionary origin is not fully understood. Our results show that the enzymes involved in these two pathways operate in the cytoplasm with the exception of the mitochondrially targeted 3-oxoadipate CoA-transferase (Osc1p) and 3-oxoadipyl-CoA thiolase (Oct1p) catalyzing the last two reactions of the 3-oxoadipate pathway. The cellular localization of the enzymes indicates that degradation of hydroxyaromatic compounds requires a shuttling of intermediates, cofactors, and products of the corresponding biochemical reactions between cytosol and mitochondria. Indeed, we found that yeast cells assimilating hydroxybenzoates increase the expression of genes SFC1, LEU5, YHM2, and MPC1 coding for succinate/fumarate carrier, coenzyme A carrier, oxoglutarate/citrate carrier, and the subunit of pyruvate carrier, respectively. A phylogenetic analysis uncovered distinct evolutionary trajectories for sparsely distributed gene clusters coding for enzymes of both pathways. Whereas the 3-oxoadipate pathway appears to have evolved by vertical descent combined with multiple losses, the gentisate pathway shows a striking pattern suggestive of horizontal gene transfer to the evolutionarily distant Mucorales.This work was supported by the Slovak grant agency (VEGA) (1/0333/15 and 1/0052/16) and the Slovak Research and Development Agency (APVV) (14-0253 and 15-0022). The T.G. group is supported in part by a grant from the Spanish ministry of Economy and Competitiveness (BIO2012-37161), a grant from the Qatar National Research Fund (NPRP 5-298-3-086), and a grant from the European Research Council (ERC) under the European Union’s Seventh Framework Program (FP/2007-2013)/ERC (grant agreement no. ERC-2012-StG-310325)

Eukaryotic transporters for hydroxyderivatives of benzoic acid

Several yeast species catabolize hydroxyderivatives of benzoic acid. However, the nature of carriers responsible for transport of these compounds across the plasma membrane is currently unknown. In this study, we analyzed a family of genes coding for permeases belonging to the major facilitator superfamily (MFS) in the pathogenic yeast Candida parapsilosis. Our results revealed that these transporters are functionally equivalent to bacterial aromatic acid: H+ symporters (AAHS) such as GenK, MhbT and PcaK. We demonstrate that the genes HBT1 and HBT2 encoding putative transporters are highly upregulated in C. parapsilosis cells assimilating hydroxybenzoate substrates and the corresponding proteins reside in the plasma membrane. Phenotypic analyses of knockout mutants and hydroxybenzoate uptake assays provide compelling evidence that the permeases Hbt1 and Hbt2 transport the substrates that are metabolized via the gentisate (3-hydroxybenzoate, gentisate) and 3-oxoadipate pathway (4-hydroxybenzoate, 2,4-dihydroxybenzoate and protocatechuate), respectively. Our data support the hypothesis that the carriers belong to the AAHS family of MFS transporters. Phylogenetic analyses revealed that the orthologs of Hbt permeases are widespread in the subphylum Pezizomycotina, but have a sparse distribution among Saccharomycotina lineages. Moreover, these analyses shed additional light on the evolution of biochemical pathways involved in the catabolic degradation of hydroxyaromatic compounds.We would like to thank Ladislav Kováč and Jordan Kolarov (Comenius University in Bratislava) for long-term support, Peter Polčic and our lab members for discussions. This work was supported by the Slovak grant agencies VEGA (1/0333/15 and 1/0052/16) and APVV (14-0253 and 15-0022) and the Comenius University grant (UK/429/2015). TG was supported in part by a grant from the Spanish Ministry of Economy and Competitiveness grants, 'Centro de Excelencia Severo Ochoa 2013-2017' SEV-2012-0208, and BFU2015-67107 cofounded by European Regional Development Fund (ERDF); from the European Union and ERC Seventh Framework Programme (FP7/2007-2013) under grant agreements FP7-PEOPLE-2013-ITN-606786 and ERC-2012-StG-310325; from the Catalan Research Agency (AGAUR) SGR857, and grant from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No H2020-MSCA-ITN-2014-642095. AG was funded by NKFIH NN 113153, by GINOP 2.3.2-15-2016-00035 and by GINOP 2.3.3-15-2016-00006