First evidence for substrate channeling between proline catabolic enzymes: A validation of domain fusion analysis for predicting protein-protein interactions

Nikhilesh Sanyal, Benjamin W. Arentson, Min Luo, John J. Tanner, Donald F Becker

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Proline dehydrogenase (PRODH) and Δ1-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, PRODHandP5CDHare fused together in the bifunctional enzyme proline utilizationA(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 PRODHand P5CDH from Thermus thermophilus are consistent with a substrate channeling mechanism, as the approach to steady-state formation of NADHdoes not fit a nonchanneling 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 PRODHandP5CDH 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 monofunctionalPRODH-P5CDH enzyme pairs.

Original languageEnglish (US)
Pages (from-to)2225-2234
Number of pages10
JournalJournal of Biological Chemistry
Volume290
Issue number4
DOIs
StatePublished - Jan 23 2015

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Proline Oxidase
Proline
Oxidoreductases
Fusion reactions
Substrates
Enzymes
Proteins
Thermus thermophilus
1-Pyrroline-5-Carboxylate Dehydrogenase
Glutamic Acid
Surface Plasmon Resonance
Structural Models
Surface plasmon resonance
Gram-Negative Bacteria
NAD
Assays
Bacteria

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

First evidence for substrate channeling between proline catabolic enzymes : A validation of domain fusion analysis for predicting protein-protein interactions. / Sanyal, Nikhilesh; Arentson, Benjamin W.; Luo, Min; Tanner, John J.; Becker, Donald F.

In: Journal of Biological Chemistry, Vol. 290, No. 4, 23.01.2015, p. 2225-2234.

Research output: Contribution to journalArticle

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abstract = "Proline dehydrogenase (PRODH) and Δ1-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, PRODHandP5CDHare fused together in the bifunctional enzyme proline utilizationA(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 PRODHand P5CDH from Thermus thermophilus are consistent with a substrate channeling mechanism, as the approach to steady-state formation of NADHdoes not fit a nonchanneling 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 PRODHandP5CDH 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 monofunctionalPRODH-P5CDH enzyme pairs.",
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