Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenaseand ∆\u3csup\u3e1\u3c/sup\u3e-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline UtilizationA(PutA)

Background: PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism. Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step. Conclusion: Substrate channeling in PutA exhibits hysteretic behavior. Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenaseand ∆\u3csup\u3e1\u3c/sup\u3e-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline UtilizationA(PutA)

Background: PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism. Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step. Conclusion: Substrate channeling in PutA exhibits hysteretic behavior. Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation

Investigation of structure-function relationships in the bifunctional PutA enzyme and the role of proline in modulating the redox environment

Proline oxidation to glutamate is catalyzed by the flavoenzyme PutA in several microorganisms. In certain prokaryotes such as Escherichia coli, PutA is also a transcriptional repressor of the proline utilization (put) genes and thus is trifunctional. We have begun to assess differences between bifunctional enzymes from Bradyrhizobium japonicum (BjPutA) and Helicobacter hepaticus (HhPutA) and the trifunctional enzyme from Escherichia coli (EcPutA). The bifunctional enzymatic activity is shared among all PutA enzymes with interesting differences attributing to the new properties exhibited by BjPutA and HhPutA. 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. This situation 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. Limited proteolysis of BjPutA under reducing conditions shows FAD reduction does not influence BjPutA conformation suggesting that the redox dependent regulation observed with EcPutA may be limited to trifunctional PutA homologues. The possibility of substrate channeling in proline catabolism was tested in 2 BjPutA. Steady-state and stopped-flow kinetic measurements provide clear evidence for a channeling mechanism in BjPutA. ^ The characterization of HhPutA showed that it exhibits about 100-fold higher oxidase activity than EcPutA. The significance of increased oxygen reactivity in HhPutA was studied by oxidative stress studies in E. coli. To understand the physiological relevance of proline metabolism, a mutant strain of H. hepaticus was generated by deleting the putA gene. From a mouse colonization studies it is apparent that PutA is imperative in causing increased inflammation and contributes to the pathology of H. hepaticus infection. ^ Proline metabolism has been associated with a number of different ecological niches, with proline having multifaceted roles in protein chaperoning, abiotic stress protection, and energy utilization. A less understood property of proline is its ability to scavenge free radicals in mammals. The potential of proline to suppress ROS levels and apoptosis in mammalian cells was studied. Proline was effective in rescuing cells from H2O2 mediated apoptosis. In cells exposed to oxidative stress, proline biosynthesis was upregulated. The proline catabolic enzyme (PRODH) was purified and showed to generate ROS as a consequence of substrate reduction of the FAD cofactor. These results suggest that proline uniquely impacts the intracellular redox environment by opposing mechanisms.

Oxygen Reactivity of PutA from Helicobacter Species and Proline-Linked Oxidative Stress

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 Δ(1)-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 (PutA(Hh)) and Helicobacter pylori (PutA(Hp)) to provide new insights into proline metabolism in these gastrointestinal pathogens. Both PutA(Hh) and PutA(Hp) lack DNA binding activity, in contrast to PutA from Escherichia coli (PutA(Ec)), which both regulates and catalyzes proline utilization. PutA(Hh) and PutA(Hp) display catalytic activities similar to that of PutA(Ec) but have higher oxygen reactivity. PutA(Hh) and PutA(Hp) exhibit 100-fold-higher turnover numbers (∼30 min(−1)) than PutA(Ec) (<0. 3 min(−1)) using oxygen as an electron acceptor during catalytic turnover with proline. Consistent with increased oxygen reactivity, PutA(Hh) forms a reversible FAD-sulfite adduct. The significance of increased oxygen reactivity in PutA(Hh) and PutA(Hp) was probed by oxidative stress studies in E. coli. Expression of PutA(Ec) and PutA from Bradyrhizobium japonicum, which exhibit low oxygen reactivity, does not diminish stress survival rates of E. coli cell cultures. In contrast, PutA(Hp) and PutA(Hh) 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

Anxious moments for the protein tyrosine phosphatase PTP1B

Chronic stress can lead to the development of anxiety and mood disorders. Thus, novel therapies for preventing adverse effects of stress are vitally important. Recently, the protein tyrosine phosphatase PTP1B was identified as a novel regulator of stress-induced anxiety. This opens up exciting opportunities to exploit PTP1B inhibitors as anxiolytics

Effect of Farnesol on a Mouse Model of Systemic Candidiasis, Determined by Use of a DPP3 Knockout Mutant of \u3ci\u3eCandida albicans\u3c/i\u3e

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 quorumsensing 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 \u3c 0.03). Similarly, mice (n = 40) injected with 1.0 ml of 20 mM farnesol i.p. had enhanced mortality (P \u3c 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 \u3c 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

Characterization of a Helicobacter hepaticus putA Mutant Strain in Host Colonization and Oxidative Stress ▿ †

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

Effect of Farnesol on a Mouse Model of Systemic Candidiasis, Determined by Use of a DPP3 Knockout Mutant of Candida albicans

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