title: First evidence for substrate channeling between proline catabolic enzymes: a validation of domain fusion analysis for predicting protein-protein interactions.
abstract: Proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) catalyze the four-electron oxidation of proline to glutamate via the intermediates P5C and l-glutamate-γ-semialdehyde (GSA). In Gram-negative bacteria, PRODH and P5CDH are fused together in the bifunctional enzyme proline utilization A (PutA) whereas in other organisms PRODH and P5CDH are expressed as separate monofunctional enzymes. Substrate channeling has previously been shown for bifunctional PutAs, but whether the monofunctional enzymes utilize an analogous channeling mechanism has not been examined. Here, we report the first evidence of substrate channeling in a PRODH-P5CDH two-enzyme pair. Kinetic data for the coupled reaction of PRODH and P5CDH from Thermus thermophilus are consistent with a substrate channeling mechanism, as the approach to steady-state formation of NADH does not fit a non-channeling two-enzyme model. Furthermore, inactive P5CDH and PRODH mutants inhibit NADH production and increase trapping of the P5C intermediate in coupled assays of wild-type PRODH-P5CDH enzyme pairs, indicating that the mutants disrupt PRODH-P5CDH channeling interactions. A dissociation constant of 3 μm was estimated for a putative PRODH-P5CDH complex by surface plasmon resonance (SPR). Interestingly, P5CDH binding to PRODH was only observed when PRODH was immobilized with the top face of its (βα)8 barrel exposed. Using the known x-ray crystal structures of PRODH and P5CDH from T. thermophilus, a model was built for a proposed PRODH-P5CDH enzyme channeling complex. The structural model predicts that the core channeling pathway of bifunctional PutA enzymes is conserved in monofunctional PRODH-P5CDH enzyme pairs.
authors.affiliation: From the Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 and.
authors.forename: Nikhilesh
authors.initials: N
authors.lastname: Sanyal
authors.affiliation: From the Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 and.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Arentson
authors.affiliation: Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211.
authors.forename: Min
authors.initials: M
authors.lastname: Luo
authors.affiliation: Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211 the Departments of Biochemistry and.
authors.forename: John J
authors.initials: JJ
authors.lastname: Tanner
authors.affiliation: From the Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 and dbecker3@unl.edu.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Becker
mesh_terms: D050842:1-Pyrroline-5-Carboxylate Dehydrogenase / Q000378:metabolism*; D002384:Catalysis; D005420:Flavoproteins / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009243:NAD / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D011392:Proline / Q000737:chemistry*; D011394:Proline Oxidase / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D005420:Flavoproteins; D009243:NAD; D011392:Proline; D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D011394:Proline Oxidase; D010100:Oxygen
keywords: Amino Acid; Bacterial Metabolism; Dehydrogenase; Enzyme Kinetics; Enzyme Mechanism; Flavoprotein; Proline; Substrate Channeling