PMID- 27742866
OWN - NLM
STAT- MEDLINE
DCOM- 20171205
LR  - 20240324
IS  - 1573-4935 (Electronic)
IS  - 0144-8463 (Print)
IS  - 0144-8463 (Linking)
VI  - 36
IP  - 6
DP  - 2016 Dec
TI  - Engineering a trifunctional proline utilization A chimaera by fusing a 
      DNA-binding domain to a bifunctional PutA.
LID - 10.1042/BSR20160435 [doi]
LID - e00413
AB  - Proline utilization A (PutA) is a bifunctional flavoenzyme with proline 
      dehydrogenase (PRODH) and Delta(1)-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.
CI  - (c) 2016 The Author(s).
FAU - Arentson, Benjamin W
AU  - Arentson BW
AD  - Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, 
      Lincoln, NE 68588, U.S.A.
FAU - Hayes, Erin L
AU  - Hayes EL
AD  - Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, 
      Lincoln, NE 68588, U.S.A.
FAU - Zhu, Weidong
AU  - Zhu W
AD  - Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, 
      Lincoln, NE 68588, U.S.A.
FAU - Singh, Harkewal
AU  - Singh H
AD  - Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, 
      U.S.A.
AD  - Protein Technologies and Assays, Research and Development, MilliporeSigma, 2909 
      Laclede Avenue, St. Louis, MO 63103, U.S.A.
FAU - Tanner, John J
AU  - Tanner JJ
AD  - Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, 
      U.S.A.
AD  - Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, 
      U.S.A.
FAU - Becker, Donald F
AU  - Becker DF
AD  - Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, 
      Lincoln, NE 68588, U.S.A. dbecker3@unl.edu.
LA  - eng
GR  - P30 GM103335/GM/NIGMS NIH HHS/United States
GR  - R01 GM061068/GM/NIGMS NIH HHS/United States
PT  - Journal Article
DEP - 20161122
PL  - England
TA  - Biosci Rep
JT  - Bioscience reports
JID - 8102797
RN  - 0 (Bacterial Proteins)
RN  - 0 (DNA-Binding Proteins)
RN  - 0 (Membrane Proteins)
RN  - 0 (PutA protein, Bacteria)
RN  - 0 (Pyrroles)
RN  - 2906-39-0 (delta-1-pyrroline-5-carboxylate)
RN  - 9DLQ4CIU6V (Proline)
RN  - EC 1.5.3.- (Proline Oxidase)
SB  - IM
MH  - Bacterial Proteins/*genetics/metabolism
MH  - Chimera/*genetics/metabolism
MH  - DNA-Binding Proteins/genetics
MH  - Escherichia coli/genetics/metabolism
MH  - Membrane Proteins/*genetics/metabolism
MH  - Proline/*genetics
MH  - Proline Oxidase/genetics
MH  - Protein Binding/genetics
MH  - Protein Domains/*genetics
MH  - Pyrroles/metabolism
MH  - Rhodobacter capsulatus/genetics
PMC - PMC5293562
OTO - NOTNLM
OT  - DNA binding
OT  - flavin enzyme
OT  - proline metabolism
OT  - small-angle X-ray scattering (SAXS)
EDAT- 2016/10/16 06:00
MHDA- 2017/12/06 06:00
PMCR- 2016/12/01
CRDT- 2016/10/16 06:00
PHST- 2016/09/29 00:00 [received]
PHST- 2016/10/05 00:00 [revised]
PHST- 2016/10/14 00:00 [accepted]
PHST- 2016/10/16 06:00 [pubmed]
PHST- 2017/12/06 06:00 [medline]
PHST- 2016/10/16 06:00 [entrez]
PHST- 2016/12/01 00:00 [pmc-release]
AID - BSR20160435 [pii]
AID - e00413 [pii]
AID - 10.1042/BSR20160435 [doi]
PST - epublish
SO  - Biosci Rep. 2016 Nov 22;36(6):e00413. doi: 10.1042/BSR20160435. Print 2016 Dec.