**6. Amino Acid Metabolism in** *C. albicans*

Many amino acids, derived either from extracellular uptake or oligopeptide/protein degradation or released from the vacuolar compartment, are converted to glutamate in the cytosol via the catalytic activity of distinct aminotransferases (ATs) (Figure 5). Specifically, ATs transfer the α-amino group of an amino acid to α-ketoglutarate (α-KG; 2-oxoglutarate), resulting in the formation of glutamate. ATs are defined by the amino acid that serves as the amino group donor. ATs collectively contribute to the cytosolic glutamate pool (Glucyto). Examples include aspartate aminotransferase (Aat1, EC 2.6.1.1), which transfers the α-amino group of aspartate to α-KG, resulting in glutamate and oxaloacetate; ornithine transaminase (Car2; EC 2.6.1.13), which uses ornithine, forming glutamate and glutamyl-5-semialdehyde; alanine transaminase (Alt1; EC 2.6.1.2), which uses alanine, forming glutamate and pyruvate; and glutamate synthase (Glt1; EC 1.4.1.14), which uses glutamine, forming glutamate.

Glutamate is enzymatically converted to α-KG via oxidative deamination, catalyzed by NAD+-dependent glutamate dehydrogenase (Gdh2; EC 1.4.1.2), yielding ammonia (NH3) and reduced NADH [61,93]. At a physiologically relevant pH, ammonia is protonated to ammonium (NH4 +). In mammalian cells, glutamate dehydrogenase is localized in the mitochondria, whereas in *S. cerevisiae,* there is a lack of consensus regarding its localization; Gdh2 has been reported to be a mitochondrial component [94,95] and alternatively a cytosolic component [96–98]. The bulk of ammonia produced by *C. albicans* during growth on amino acids as a sole nitrogen and carbon source comes from the reaction catalyzed by Gdh2 [61]. Contrary to the initial publication, now corrected [61], Gdh2 in *C. albicans* is clearly cytoplasmic. The correct assignment of Gdh2 as a cytoplasmic component is key to understanding its role in cellular energy production, as the reaction reduces NAD+, forming NADH. The ammonium pool in the cytosol generated primarily from Gdh2 activity and possibly from the import of extracellular ammonium via the ammonium transporters Mep1 and Mep2 [57,99,100] can be assimilated by two key anabolic reactions catalyzed by the NADPH-dependent glutamate dehydrogenase (Gdh3, EC 1.4.1.4; Gdh1 in *S. cerevisiae)* and glutamine synthetase (Gln1; EC 6.3.1.2) (Figure 5). Gdh3 catalyzes the synthesis of glutamate from α-KG and ammonium [93], whereas Gln1 catalyzes the synthesis of glutamine from glutamate and ammonium in an ATP-dependent reaction [93]. In *S. cerevisiae*, cytosolic glutamate can be imported to the mitochondrial matrix via transporters localized at the inner mitochondrial membrane, such as Agc1 [101–104] or Ymc2 [104]. Putative orthologs of these proteins exist in *C. albicans* (see CGD, http://www.candidagenome.org (accessed

on 19 December 2021); C1\_13400C for Agc1 and C4\_02080W for Ymc2). Furthermore, in *S. cerevisiae*, cytosolic α-KG can be imported into the mitochondria through oxodicarboxylate carriers that exists in two isoforms, Odc1 and Odc2; Odc1 is used during respiration and its expression is subject to glucose repression, whereas Odc2 is the predominant isoform under non-respiratory conditions [105,106]. The *C. albicans* genome has a putative ortholog for Odc1 (CR\_05480W).

**Figure 5. Nitrogen utilization in** *C. albicans***.** Most free amino acids, obtained from extracellular uptake (**1**), vacuolar release (**2**) or the degradation of small peptides by intracellular peptidases (**3**), are deaminated by specific aminotransferases (ATs) using α-ketoglutarate as the amino group acceptor, forming glutamate. Many ATs are found in the cytosol and there is an abundant supply of glutamate in the cytosol (Glucyto) (**4**). Glutamate can be imported into the mitochondria by transporters, such as Agc1 (**5**). Arginine and proline are converted to glutamate via the proline catabolic pathway (**6**). In the cytosolic portion of this pathway, arginine is converted to ornithine and urea by arginase (Car1). Ornithine is rapidly converted to glutamate semialdehyde (GSA) by ornithine transaminase (Car2), which is non-enzymatically converted to the cyclic Δ-1-pyrroline-5-carboxylate (P5C) and then reduced by P5C reductase (Pro3), generating proline (Procyto). Promito) via an unidentified transporter. Promito is catabolized to glutamate (Glumito) via the concerted activities of proline dehydrogenase (Put1) and P5C dehydrogenase (Put2).

Proline enters the mitochondria (Glutamate produced in the mitochondria (Glumito) (**8**) is thought to exit the mitochondria and become part of Glucyto. Glucyto is used in the synthesis of proline (**7**); glutamate is first activated to produce glutamate-5-phosphate (G5P) by γ-glutamyl kinase (Pro1), followed by its conversion to GSA/P5C by γ-glutamyl phosphate reductase (Pro2) and is then reduced to proline by P5C reductase (Pro3). Cytosolic glutamate can be converted to α-ketoglutarate by the NAD+-dependent glutamate dehydrogenase (Gdh2), which is critical for maintaining the α-ketoglutarate pool in the cytosol (α-KGcyto). The reaction catalyzed by Gdh2 generates ammonia as a by-product (**9**). α-ketoglutarate can be transported in and out of mitochondria via putative oxodicarboxylate carriers (e.g., Odc1). The mitochondrial α-KGmito pool is linked to the TCA cycle (**10**). Urea, derived either from arginine or from extracellular uptake, can be converted to ammonia and CO2 via urea amidolyase (Dur1,2) (**11**). When grown in the presence of N-acetylglucosamine (GlcNac), ammonia is also produced when glucosamine-6-phosphate is converted to fructose-6-phosphate through glucosamine-6-phosphate isomerase (Nag1) (**12**). As the cytosolic pH is maintained near neutrality (pH~6.5), most ammonia is converted to its protonated form, ammonium (**13**). Free ammonia is membrane-permeable and can readily exit cells, where it contributes to the alkalization of the growth environment, a consequence of its conversion to ammonium (**14**). Ammonium in cells can be reassimilated to generate glutamate by the NADPH-dependent glutamate dehydrogenase (Gdh3), which uses α-ketoglutarate as a substrate (**15**); additionally, ammonium can be reassimilated via glutamine synthetase (Gln1), which catalyzes the conversion of glutamate to glutamine (**16**). Glutamate can also be generated from glutamine and α-ketoglutarate via the NADH-dependent glutamate synthase (Glt1) (**17**). In mitochondria, the NADH/FADH2 generated by the TCA cycle and proline catabolism can be oxidized via the electron transport chain (ETC) to generate ATP (**18**). Mitochondrial function and multiple enzymatic activities are repressed in cells grown in the presence of high glucose (≥0.2%, (**19**)). The localization of enzymes shown in green have been experimentally validated in *C. albicans*, whereas those shown in red are based on the localization of their corresponding orthologs in *S. cerevisiae* and the presence or absence of strong mitochondrial pre-sequences in the N-terminals of their respective protein sequences (Candida Genome Database (CGD, http://www.candidagenome.org (accessed on 18 December 2021)) analyzed using the MitoFate tool [92]. The following enzymes are present in the *C. albicans* genome—PRODH = *PUT1* (C5\_02600W), P5CDH = *PUT2* (C5\_04880C), GDH = *GDH2* (C2\_07900W), P5CR = *PRO3* (C4\_00240), OAT = *CAR2* (C4\_00160C), ARG = *CAR1* (C5\_04490C), GK = *PRO1* (CR\_10580), GPR = *PRO2* (C3\_07220C). *GLN1* (CR\_05050W), *GLT1* (C1\_06550W), *DUR1,2* (C1\_04660W), NADPH-dependent GDH = *GDH3* (C4\_06120W), *AAT1* (C2\_05250C) and *AAT2* = *AAT21* (CR\_07620W).

In addition to the cytoplasmic glutamate pool (Glucyto), a significant fraction of the intracellular glutamate is generated in the mitochondria (Glumito) via the proline catabolic pathway. In eukaryotes, the four-electron catabolic conversion of proline to glutamate is carried out through the successive actions of proline dehydrogenase (PRODH; EC 1.5.5.2) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH; EC 1.2.1.88). PRODH and P5CDH are highly conserved enzymes throughout eukaryotes and bacteria (reviewed in [107–110]). In *C. albicans*, PRODH and P5CDH are called Put1 and Put2, respectively, and both are nuclear-encoded mitochondrial proteins [59,61]; in most eukaryotes, PRODH is associated with the inner mitochondrial membrane and is connected to complex II of the electron transport chain (ETC; reviewed in [110]). Cytosolic proline (Procyto), derived from uptake, biosynthetic reactions or from the catabolism of arginine or ornithine [59], is imported into the mitochondria (Promito) via a still-unidentified mitochondrial transporter. PRODH then transfers two electrons from proline to FAD to generate P5C and the reduced flavin cofactor (FADH2). P5C tautomerizes spontaneously in a non-enzymatic reaction, forming glutamic-γ-semialdehyde (GSA). The prevailing pH strongly affects the equilibrium between P5C and GSA; P5C formation is favored when the pH is >6.5. P5CDH then catalyzes the oxidation of GSA to glutamate, reducing NAD+ to NADH. When high levels of proline are available and catabolized, P5C can accumulate in the mitochondria and exert a toxic effect [111–113]. The reduced cofactors FADH2 and NADH, generated by proline catabolism, are oxidized by the ETC of mitochondria to power ATP generation. Since Gdh2

catalyzes the conversion of glutamate to α-KG in the cytosol and that Gdh2-dependent alkalization is tightly linked to mitochondrial function [61], it is highly likely that glutamate resulting from proline catabolism (Glumito) is able to exit the mitochondria. To date, a dedicated glutamate transporter capable of exporting glutamate out of the mitochondria has yet to be identified. In yeast, mitochondrial glutamate is converted to aspartate by the mitochondrial aspartate aminotransferase (Aat1; EC 2.6.1.1), and aspartate exits the mitochondria via the Agc1 antiporter. The relevance of this transporter with respect to the export of glutamate is not clear, as the antiporter mechanism transports aspartate out and glutamate in. Aspartate in the cytosol can be converted back to glutamate via the cytosolic aspartate aminotransferase (Aat2; EC 2.6.1.1). The *C. albicans* genome has putative orthologs of Aat1 (*AAT1*; C2\_05250C) and Aat2 (*AAT21*; CR\_07620W). In yeast, cytosolic glutamate is used in the biosynthesis of several amino acids, including proline; 85% of the total cellular nitrogen is incorporated via the amino nitrogen of glutamate, and the remaining 15% is derived from the amide nitrogen of glutamine [114].
