Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA

Arentson, Benjamin W.; Hayes, Erin L.; Zhu, Weidong; Singh, Harkewal; Tanner, John J.; Becker, Donald F.

doi:10.1042/bsr20160435

Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA

Journal Article · Tue Nov 22 00:00:00 EST 2016 · Bioscience Reports

DOI:https://doi.org/10.1042/bsr20160435· OSTI ID:1624992

Arentson, Benjamin W. ^[1]; Hayes, Erin L. ^[2]; Zhu, Weidong ^[2]; Singh, Harkewal ^[3]; Tanner, John J. ^[4]; Becker, Donald F. ^[2]

Univ. of Nebraska, Lincoln, NE (United States). Redox Biology Center. Dept. of Biochemistry; DOE/OSTI
Univ. of Nebraska, Lincoln, NE (United States). Redox Biology Center. Dept. of Biochemistry
Univ. of Missouri, Columbia, MO (United States). Dept. of Chemistry; MilliporeSigma, St. Louis, MO (United States). Research and Development. Protein Technologies and Assays
Univ. of Missouri, Columbia, MO (United States). Dept. of Biochemistry; Univ. of Missouri, Columbia, MO (United States). Dept. of Chemistry

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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1624992

Journal Information:: Bioscience Reports, Journal Name: Bioscience Reports Journal Issue: 6 Vol. 36; ISSN 0144-8463

Publisher:: Portland Press - Biochemical SocietyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA

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