First Evidence for Substrate Channeling between Proline Catabolic Enzymes \u3ci\u3eA VALIDATION OF DOMAIN FUSION ANALYSIS FOR PREDICTING PROTEIN-PROTEIN INTERACTIONS\u3c/i\u3e

Background: PRODH and P5CDH from Thermus thermophilus are monofunctional enzymes in proline catabolism. Results: Steady-state kinetics and intermediate trapping data show the PRODH and P5CDH reactions are coupled by a channeling step. Conclusion: Substrate channeling in monofunctional enzymes is achieved via weak interactions. Significance: Evidence for substrate channeling between monofunctional proline catabolic enzymes is shown and confirms the Rosetta Stone hypothesis

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

First Evidence for Substrate Channeling between Proline Catabolic Enzymes \u3ci\u3eA VALIDATION OF DOMAIN FUSION ANALYSIS FOR PREDICTING PROTEIN-PROTEIN INTERACTIONS\u3c/i\u3e

Background: PRODH and P5CDH from Thermus thermophilus are monofunctional enzymes in proline catabolism. Results: Steady-state kinetics and intermediate trapping data show the PRODH and P5CDH reactions are coupled by a channeling step. Conclusion: Substrate channeling in monofunctional enzymes is achieved via weak interactions. Significance: Evidence for substrate channeling between monofunctional proline catabolic enzymes is shown and confirms the Rosetta Stone hypothesis

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

Mystify me: Coke, terror and the symbolic immortality boost

A panel on “Marketing as Mystification” convened at the 2011 Academy of Marketing conference in Liverpool. Ideas from the Liverpool event were supplemented by commentaries from selected other authors. Each commentary explores the aspects of “mystification” observable in marketing discourses and practices. In what follows, Laufer interprets marketing mystification as modern form of sophism, Dholakia and Firat discuss mystifying ways that inequality is marketed, Varman analyzes the perversion and mystification of “development” via neoliberal marketing of “social entrepreneurship,” Mikkonen explores mystifying marketing representations of gays and lesbians, and Freund and Jacobi present a fascinating interpretation of how Coca-Cola advertising mystically reassures us that our difficult, dangerous lifeworld is actually quite hunky-dory. </jats:p

Investigations of Substrate Channeling in the Proline Oxidative Pathway

In cell metabolism, substrate channeling is a phenomenon where the product of one reaction is transported to a second enzyme active site without equilibrating into bulk solvent. Chapter 1 reviews the rationale and evidence for substrate channeling with the specific example of proline metabolism. Oxidation of proline to glutamate is catalyzed in consecutive reactions by proline dehydrogenase (PRODH) and pyrroline-5-carboxylate dehydrogenase (P5CDH). The intermediate Δ1-pyrroline-5-carboxylate reportedly tends to be labile and inhibitory towards several metabolic pathways. One of the main objectives of this dissertation was to investigate substrate channeling between independent proline oxidative enzymes from Thermus thermophilus- TtPRODH and TtP5CDH. Chapter 2 establishes that TtPRODH and TtP5CDH are capable of interacting with a dissociation constant (KD) of 3.03 µM as demonstrated using Surface Plasmon Resonance (SPR). As observed in the present study, this interaction is possible only with a specific orientation of TtPRODH relative to TtP5CDH. A docking model of the two enzymes predicts an orientation of the active sites which is supportive of substrate channeling. Corroborating observations are made with kinetic studies. We observe that interference of TtPRODH-TtP5CDH complex by catalytically inactive mutants TtPRODH R288M/R289M and TtP5CDH C322A lead to significant decrease in glutamate formation. The results pave the way for testing substrate channeling in eukaryotic enzymes. In chapter 3, two novel eukaryotic enzymes from Saccharomyces cerevisiae, Put1p (PRODH) and Put2p (P5CDH), have been characterized. Particular attention was focused on the oxidative half-reaction of Put1p for gaining insight into possible redox functions of human PRODH. Previous studies show that bifunctional enzyme from Gram-negative Bradyrhizobium japonicum (BjPutA) containing PRODH and P5CDH domains, exhibits substrate channeling via an elegant internal tunnel. BjPutA and its channeling variants were used to test the role of substrate channel in hydrolysis of P5C, an essential step in proline oxidation. These aspects of substrate channeling are discussed in chapter 4. Overall, this study provides an improved understanding of: (1) Substrate channeling in proline oxidation; and (2) a model for investigating substrate channeling between other individual enzymes that catalyze consecutive reactions. Advisor: Donald F. Becke

Substrate channeling in proline metabolism

Proline metabolism is an important pathway that has relevance in several cellular functions such as redox balance, apoptosis, and cell survival. Results from different groups have indicated that substrate channeling of proline metabolic intermediates may be a critical mechanism. One intermediate is pyrroline-5-carboxylate (P5C), which upon hydrolysis opens to glutamic semialdehyde (GSA). Recent structural and kinetic evidence indicate substrate channeling of P5C/ GSA occurs in the proline catabolic pathway between the proline dehydrogenase and P5C dehydrogenase active sites of bifunctional proline utilization A (PutA). Substrate channeling in PutA is proposed to facilitate the hydrolysis of P5C to GSA which is unfavorable at physiological pH. The second intermediate, gamma-glutamyl phosphate, is part of the proline biosynthetic pathway and is extremely labile. Substrate channeling of gamma-glutamyl phosphate is thought to be necessary to protect it from bulk solvent. Because of the unfavorable equilibrium of P5C/GSA and the reactivity of gamma-glutamyl phosphate, substrate channeling likely improves the efficiency of proline metabolism. Here, we outline general strategies for testing substrate channeling and review the evidence for channeling in proline metabolism

Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A

<i>Escherichia coli</i> proline utilization A (<i>Ec</i>PutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. <i>Ec</i>PutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of <i>Ec</i>PutA. Upon proline oxidation, the flavin becomes reduced, triggering a conformational change that causes <i>Ec</i>PutA to dissociate from the <i>put</i> regulon and bind to the cellular membrane. Major structure/function domains of <i>Ec</i>PutA have been characterized, including the DNA-binding domain, proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase catalytic domains, and an aldehyde dehydrogenase superfamily fold domain. Still lacking is an understanding of the membrane-binding domain, which is essential for <i>Ec</i>PutA catalytic turnover and functional switching. Here, we provide evidence for a conserved C-terminal motif (CCM) in <i>Ec</i>PutA having a critical role in membrane binding. Deletion of the CCM or replacement of hydrophobic residues with negatively charged residues within the CCM impairs <i>Ec</i>PutA functional and physical membrane association. Furthermore, cell-based transcription assays and limited proteolysis indicate that the CCM is essential for functional switching. Using fluorescence resonance energy transfer involving dansyl-labeled liposomes, residues in the α-domain are also implicated in membrane binding. Taken together, these experiments suggest that the CCM and α-domain converge to form a membrane-binding interface near the PRODH domain. The discovery of the membrane-binding region will assist efforts to define flavin redox signaling pathways responsible for <i>Ec</i>PutA functional switching

Evidence That the C‑Terminal Domain of a Type B PutA Protein Contributes to Aldehyde Dehydrogenase Activity and Substrate Channeling

Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ1- pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress curve for the coupled PRODH−P5CDH activity of RcPutA does not exhibit a time lag, implying a substrate channeling mechanism. RcPutA is monomeric in solution, which is unprecedented for PutAs. SAXS rigid body modeling with target−decoy validation is used to build a model of RcPutA. On the basis of homology to aldehyde dehydrogenases (ALDHs), the CTDUF is predicted to consist of a β-hairpin fused to a noncatalytic Rossmann fold domain. The predicted tertiary structural interactions of the CTDUF resemble the quaternary structural interactions in the type A PutA dimer interface. The model is tested by mutagenesis of the dimerization hairpin of a type A PutA and the CTDUF hairpin of RcPutA. Similar functional phenotypes are observed in the two sets of variants, supporting the hypothesis that the CTDUF mimics the type A PutA dimer interface. These results suggest annotation of the CTDUF as an ALDH superfamily domain that facilitates P5CDH activity and substrate channeling by stabilizing the aldehyde-binding site and sealing the substrate-channeling tunnel from the bulk medium