TY - JOUR
T1 - First evidence for substrate channeling between proline catabolic enzymes
T2 - A validation of domain fusion analysis for predicting protein-protein interactions
AU - Sanyal, Nikhilesh
AU - Arentson, Benjamin W.
AU - Luo, Min
AU - Tanner, John J.
AU - Becker, Donald F.
N1 - Publisher Copyright:
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2015/1/23
Y1 - 2015/1/23
N2 - 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.
AB - 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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U2 - 10.1074/jbc.M114.625483
DO - 10.1074/jbc.M114.625483
M3 - Article
C2 - 25492892
AN - SCOPUS:84921445381
VL - 290
SP - 2225
EP - 2234
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 4
ER -