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.

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.