title: Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA.
abstract: Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon-helix-helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH-RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH-RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs.
authors.affiliation: Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE 68588, U.S.A.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Arentson
authors.affiliation: Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE 68588, U.S.A.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Hayes
authors.affiliation: Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE 68588, U.S.A.
authors.forename: Weidong
authors.initials: W
authors.lastname: Zhu
authors.affiliation: Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, U.S.A.|Protein Technologies and Assays, Research and Development, MilliporeSigma, 2909 Laclede Avenue, St. Louis, MO 63103, U.S.A.
authors.forename: Harkewal
authors.initials: H
authors.lastname: Singh
authors.affiliation: Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, U.S.A.|Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, U.S.A.
authors.forename: John J
authors.initials: JJ
authors.lastname: Tanner
authors.affiliation: Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE 68588, U.S.A. dbecker3@unl.edu.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Becker
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002678:Chimera / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D011392:Proline / Q000235:genetics*; D011394:Proline Oxidase / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D000072417:Protein Domains / Q000235:genetics*; D011758:Pyrroles / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria; D011758:Pyrroles; C015485:delta-1-pyrroline-5-carboxylate; D011392:Proline; D011394:Proline Oxidase
keywords: DNA binding; flavin enzyme; proline metabolism; small-angle X-ray scattering (SAXS)