Although originally considered toxic, hydrogen sulfide (H2S) has been implicated in mediating various biological processes. Nevertheless, its cellular targets and mode of action are not well understood. Protein Tyrosine Phosphatases (PTPs), which regulate numerous signal transduction pathways, utilize an essential Cys residue at the active site, which is characterized by a low pKa and is susceptible to reversible oxidation. Here, we report that PTP1B, the founding member of this enzyme family, was reversibly inactivated by H2S, in vitro and in vivo, via sulfhydration of the active site Cys residue. Unlike oxidized PTP1B, the sulfhydrated enzyme was preferentially reduced by thioredoxin in vitro, compared to glutathione or dithiothreitol. Sulfhydration of the active site Cys in PTP1B in cells required the presence of cystathionine-γ-lyase (CSE), a critical enzyme in H2S production, and resulted in inhibition of phosphatase activity. Suppression of CSE decreased H2S production and decreased the phosphorylation on Tyr619, and activation, of PERK [protein kinase-like endoplasmic reticulum (ER) kinase] in response to ER stress. PERK, which phosphorylates the eukaryotic translational initiation factor 2 (eIF2α) leading to attenuation of protein translation, was a direct substrate of PTP1B. In addition, CSE knockdown also led to activation of SRC, previously shown to be mediated by PTP1B. These effects of suppressing H2S production on the response to ER stress were abrogated by a small molecule inhibitor of PTP1B. Together, these data define a signaling function for H2S in inhibiting PTP1B activity and thereby promoting PERK activity during the response to ER stress.

Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria, but are fused into bifunctional enzymes known as Proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0 Å resolution structure of Thermus thermophilus PRODH reveals a distorted (βα)8 barrel catalytic core domain and a hydrophobic α-helical domain located above the carboxyl terminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent exposed compared to PutA due to a 4-Å shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify 9 conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is −75 mV and the kinetic parameters for proline are Km=27 mM and kcat=13 s−1. 3,4-dehydro-L-proline was found to be an efficient substrate and L-tetrahydro-2-furoic acid is a competitive inhibitor (KI=1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O2 producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs.

The potential of proline to suppress reactive oxygen species (ROS) and apoptosis in mammalian cells was tested by manipulating intracellular proline levels exogenously and endogenously by overexpression of proline metabolic enzymes. Proline was observed to protect cells against H2O2, tert-butyl hydroperoxide and a carcinogenic oxidative stress inducer but was not effective against superoxide generators such as menadione. Oxidative stress protection by proline requires the secondary amine of the pyrrolidine ring and involves preservation of the glutathione redox environment. Overexpression of proline dehydrogenase (PRODH), a mitochondrial flavoenzyme that oxidizes proline, resulted in 6-fold lower intracellular proline content and decreased cell survival relative to control cells. Cells overexpressing PRODH were rescued by pipecolate, an analog that mimics the antioxidant properties of proline, and by tetrahydro-2-furoic acid, a specific inhibitor of PRODH. In contrast, overexpression of the proline biosynthetic enzymes Δ1-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) resulted in 2-fold higher proline content, significantly lower ROS levels and increased cell survival relative to control cells. In different mammalian cell lines exposed to physiological H2O2 levels, increased endogenous P5CS and P5CR expression was observed indicating upregulation of proline biosynthesis is an oxidative stress response.

Helicobacter hepaticus is a gram-negative, spiral-shaped microaerophilic bacterium associated with chronic intestinal infection leading to hepatitis and colonic and hepatic carcinomas in susceptible strains of mice. In the closely related human pathogen Helicobacter pylori, l-proline is a preferred respiratory substrate and is found at significantly high levels in the gastric juice of infected patients. A previous study of the proline catabolic PutA flavoenzymes from H. pylori and H. hepaticus revealed that Helicobacter PutA generates reactive oxygen species during proline oxidation by transferring electrons from reduced flavin to molecular oxygen. We further explored the preference for proline as a respiratory substrate and the potential impact of proline metabolism on the redox environment in Helicobacter species during host infection by disrupting the putA gene in H. hepaticus. The resulting putA knockout mutant strain was characterized by oxidative stress analysis and mouse infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to that of the wild-type strain, consistent with proline's role as an antioxidant. The significant increase in stress resistance was attributed to higher proline content, as no upregulation of antioxidant genes was observed for the putA mutant strain. The wild-type and putA mutant H. hepaticus strains displayed similar levels of infection in mice, but in mice challenged with the putA mutant strain, significantly reduced inflammation was observed, suggesting a role for proline metabolism in H. hepaticus pathogenicity in vivo.

PutA is a bifunctional flavoenzyme in bacteria that catalyzes the four-electron oxidation of proline to glutamate. In certain prokaryotes such as Escherichia coli, PutA is also a transcriptional repressor of the proline utilization (put) genes and thus is trifunctional. In this work, we have begun to assess differences between bifunctional and trifunctional PutA enzymes by examining the PutA protein from Bradyrhizobium japonicum (BjPutA). Primary structure analysis of BjPutA shows it lacks the DNA-binding domain of E. coli PutA (EcPutA). Consistent with this prediction, purified BjPutA does not exhibit DNA-binding activity in native gel mobility shift assays with promoter regions of the putA gene from B. japonicum. The catalytic and redox properties of BjPutA were characterized and a reduction potential (Em) value of −0.132 V (pH 7.5) was determined for the bound FAD/FADH2 couple in BjPutA that is significantly more negative (∼ 55 mV) than the Em for EcPutA-bound FAD. The more negative Em value thermodynamically limits proline reduction of the FAD cofactor in BjPutA. In the presence of phospholipids, reduction of BjPutA is stimulated suggesting lipids influence the FAD redox environment. Accordingly, an Em value of −-0.114 V (pH 7.5) was determined for BjPutA-bound FAD in the presence of polar lipids. The molecular basis for the lower reduction potential of FAD in BjPutA relative to EcPutA was explored by site-directed mutagenesis. Amino acid sequence alignment between BjPutA and EcPutA indicates only one difference in active site residues near the isoalloxazine ring of FAD: Val-402 in EcPutA is substituted at the analogous position in BjPutA with Ala-310. Replacement of A310 by Val in the BjPutA mutant A310V raised the reduction potential of bound FAD relative to wild-type BjPutA to an Em value of −0.09 V (pH 7.5). The > 40-mV positive shift in the potential of the BjPutA mutant A310V suggests that the corresponding Val residue in EcPutA helps poise the FAD redox potential for thermodynamically favored proline reduction thereby allowing EcPutA to be efficiently regulated by proline availability. Limited proteolysis of BjPutA under reducing conditions shows FAD reduction does not influence BjPutA conformation indicating further that the redox dependent regulation observed with EcPutA may be limited to trifunctional PutA homologues.

This work extends our previous observation that the fungus Candida albicans secretes micromolar levels of farnesol and that accumulation of farnesol in vitro prevents the yeast-to-mycelium conversion in a quorum-sensing manner. What does farnesol do in vivo? The purpose of this study was to determine the role of farnesol during infection with a well-established mouse model of systemic candidiasis with C. albicans A72 administered by tail vein injection. This question was addressed by altering both endogenous and exogenous farnesol. For endogenous farnesol, we created a knockout mutation in DPP3, the gene encoding a phosphatase which converts farnesyl pyrophosphate to farnesol. This mutant (KWN2) produced six times less farnesol and was ca. 4.2 times less pathogenic than its SN152 parent. The strain with DPP3 reconstituted (KWN4) regained both its farnesol production levels and pathogenicity. These mutants (KWN1 to KWN4) retained their full dimorphic capability. With regard to exogenous farnesol, farnesol was administered either intraperitoneally (i.p.) or orally in the drinking water. Mice receiving C. albicans intravenously and farnesol (20 mM) orally had enhanced mortality (P < 0.03). Similarly, mice (n = 40) injected with 1.0 ml of 20 mM farnesol i.p. had enhanced mortality (P < 0.03), and the onset of mortality was 30 h sooner than for mice which received a control injection without farnesol. The effect of i.p. farnesol was more pronounced (P < 0.04) when mice were inoculated with a sublethal dose of C. albicans. These mice started to die 4 days earlier, and the percent survival on day 6 postinoculation (p.i.) was five times lower than for mice receiving C. albicans with control i.p. injections. In all experiments, mice administered farnesol alone or Tween 80 alone remained normal throughout a 14-day observation period. Finally, beginning at 12 h p.i., higher numbers of C. albicans cells were detected in kidneys from mice receiving i.p. farnesol than in those from mice receiving control i.p. injections. Thus, reduced endogenous farnesol decreased virulence, while providing exogenous farnesol increased virulence. Taken together, these data suggest that farnesol may play a role in disease pathogenesis, either directly or indirectly, and thus may represent a newly identified virulence factor.

Proline is converted to glutamate in two successive steps by the proline utilization A (PutA) flavoenzyme in gram-negative bacteria. PutA contains a proline dehydrogenase domain that catalyzes the flavin adenine dinucleotide (FAD)-dependent oxidation of proline to Δ1-pyrroline-5-carboxylate (P5C) and a P5C dehydrogenase domain that catalyzes the NAD+-dependent oxidation of P5C to glutamate. Here, we characterize PutA from Helicobacter hepaticus (PutAHh) and Helicobacter pylori (PutAHp) to provide new insights into proline metabolism in these gastrointestinal pathogens. Both PutAHh and PutAHp lack DNA binding activity, in contrast to PutA from Escherichia coli (PutAEc), which both regulates and catalyzes proline utilization. PutAHh and PutAHp display catalytic activities similar to that of PutAEc but have higher oxygen reactivity. PutAHh and PutAHp exhibit 100-fold-higher turnover numbers (∼30 min−1) than PutAEc (<0. 3 min−1) using oxygen as an electron acceptor during catalytic turnover with proline. Consistent with increased oxygen reactivity, PutAHh forms a reversible FAD-sulfite adduct. The significance of increased oxygen reactivity in PutAHh and PutAHp was probed by oxidative stress studies in E. coli. Expression of PutAEc and PutA from Bradyrhizobium japonicum, which exhibit low oxygen reactivity, does not diminish stress survival rates of E. coli cell cultures. In contrast, PutAHp and PutAHh expression dramatically reduces E. coli cell survival and is correlated with relatively lower proline levels and increased hydrogen peroxide formation. The discovery of reduced oxygen species formation by PutA suggests that proline catabolism may influence redox homeostasis in the ecological niches of these Helicobacter species.