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Revealing the function of a linker in the multi‐functional regulatory protein—proline utilization A from Escherichia coli
Author(s) -
Mao Yizi,
Arentson Benjamin W.,
Becker Donald F.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02147
Subject(s) - proline dehydrogenase , biochemistry , repressor , regulon , biology , linker , mutant , nad+ kinase , proline , escherichia coli , dehydrogenase , enzyme , amino acid , transcription factor , chemistry , gene , computer science , operating system
Proline metabolism is critical for several cellular processes such as serving as carbon and nitrogen sources, preventing osmotic stress especially in plants, scavenging reactive oxygen species, and promoting pathogen virulence. In all organisms proline is oxidized into glutamate by the coordinated activities of the flavin‐dependent enzyme L‐proline dehydrogenase (PRODH) and NAD + ‐dependent enzyme L‐glutamate‐γ‐semialdehyde (GSAL) dehydrogenase (GSALDH). In gram‐negative bacteria, these two enzymes are encoded on one polypeptide called proline utilization A (PutA). PutA from certain bacteria such as Escherichia coli (EcPutA), also includes a ribbon‐helix‐helix (RHH) DNA binding domain at the N‐terminus that is tethered to the PRODH domain by a 35 amino acid linker corresponding to residues 50–85 of the total 1320 in EcPutA. The RHH domain enables PutA to function as a transcription repressor of the put regulon ( putP and putA genes) in addition to its enzymatic roles. This study aims to understand the role of the linker in the redox regulation of PutA and its functions. As conformational changes are required for PutA to switch between transcriptional repressor and enzymatic functions, the linker is proposed to be important for optimal arrangement of the RHH and PRODH domains. Partial and total linker deletion mutants, Δ50–73 EcPutA and Δ50–85 EcPutA, respectively, were characterized. Purified Δ50–85 EcPutA was shown to behave similarly to wild‐type PutA as assessed by enzyme activity and DNA binding assays. The Δ50–85 EcPutA mutant depleted of the full linker region, however, was unable to function as a transcription repressor in cell based assays indicating that the linker is necessary for EcPutA to bind to DNA in vivo . Work is ongoing to determine whether the transcription repressor activity of EcPutA is restored in partial linker mutant Δ50–73 EcPutA. Support or Funding Information NIH grants R01GM061068 and P30GM103335. Department of Biochemistry and Redox Biology Center, University of Nebraska‐Lincoln.Model for regulation of put regulon.sDomain structure and small angle X‐ray scattering model of EcPutA. Adapted from "Structure, function, and mechanism of proline," by L. Liu, J. J. Tanner, and D. F. Becker. 2017, Archives of Biochemistry and Biophysics, 632 , p. 142. Copyright 2019 by 2019 Elsevier B.V.