Degradation of the multifunctional amino acid proline is associated with mitochondrial oxidative respiration. The two-step oxidation of proline is catalyzed by proline oxidase and D 1 -pyrroline-5-carboxylate (P5C) dehydrogenase, which produce P5C and glutamate, respectively. In animal and plant cells, impairment of P5C dehydrogenase activity results in P5C-proline cycling when exogenous proline is supplied via the actions of proline oxidase and P5C reductase (the enzyme that converts P5C to proline). This proline is oxidized by the proline oxidase-FAD complex that delivers electrons to the electron transport chain and to O 2 , leading to mitochondrial reactive oxygen species (ROS) overproduction. Coupled activity of proline oxidase and P5C dehydrogenase is therefore important for maintaining ROS homeostasis. In the genome of the fungal pathogen Cryptococcus neoformans, there are two paralogs (PUT1 and PUT5) that encode proline oxidases and a single ortholog (PUT2) that encodes P5C dehydrogenase. Transcription of all three catabolic genes is inducible by the presence of proline. However, through the creation of deletion mutants, only Put5 and Put2 were found to be required for proline utilization. The put2D mutant also generates excessive mitochondrial superoxide when exposed to proline. Intracellular accumulation of ROS is a critical feature of cell death; consistent with this fact, the put2D mutant exhibits a slight, general growth defect. Furthermore, Put2 is required for optimal production of the major cryptococcal virulence factors. During murine infection, the put2D mutant was discovered to be avirulent; this is the first report highlighting the importance of P5C dehydrogenase in enabling pathogenesis of a microorganism.P ROLINE is an important biological proteinogenic amino acid that is necessary for primary metabolism in organisms including animals, plants, fungi, and bacteria. This imino acid, as it is more precisely known, has multiple physiological functions. Apart from being an osmoprotectant, proline can serve as a transient storage of organic nitrogen, stabilize proteins and membranes during stressful conditions, prevent protein aggregation during its folding or refolding, and scavenge reactive oxygen species (ROS) such as superoxide anions (Rudolph and Crowe 1985;Carpenter and Crowe 1988;Ignatova and Gierasch 2006;Kaul et al. 2008). However, the exact mechanism by which proline protects against stresses remains poorly understood. Intracellular proline must be present at appropriate levels to confer stress-protective effects, and an excess of free proline has been shown to be detrimental to cell growth or protein functions in numerous organisms (Davis 2000;Morita et al. 2002;Deuschle et al. 2004; Maxwell and Nomura and Takagi 2004). The transport, anabolism, and catabolism of proline therefore need to be tightly regulated for proper cellular defense, adaptation, and growth.Degradation of proline that takes place in eukaryotic mitochondria or prokaryotic plasma membrane results in highener...