Proline Dehydrogenase from Escherichia coli K12

Abrahamson, John; Baker, L.; Stephenson, James T.; Wood, Janet M.

doi:10.1111/j.1432-1033.1983.tb07533.x

Cited by 68 publications

(80 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A putative regulatory DNA region (579 bp) upstream of the putA gene in B. japonicum was prepared by PCR amplification from the plasmid pPutArc using primers 5'-ACTTCCCTAGCCCACCGGCAACG-3' and 5'-CGTAGGGGGCGGTGAAGGGAGGC-3' (5). Membrane vesicles were prepared from E. coli strain JT31 putA -as described (2). E. coli polar lipid extracts and phosphatidylcholine (PC) were purchased from Avanti Polar Lipids Inc. and phospholipid suspensions were prepared as previously described (18).…”

Section: Methodsmentioning

confidence: 99%

“…The oxidation of proline is accomplished by successive FAD-and NAD-dependent steps generating glutamate (2,3,6). In the first step, a proline dehydrogenase (PRODH) domain utilizes a noncovalently bound FAD to catalyze the two-electron oxidation of proline to form 1 Δ-pyrroline-5-carboxylate (P5C).…”

mentioning

confidence: 99%

“…microbes Pseudomonas putida and Bradyrhizobium japonicum (1)(2)(3)(4)(5). The oxidation of proline is accomplished by successive FAD-and NAD-dependent steps generating glutamate (2,3,6).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Characterization of a Bifunctional PutA Homologue fromBradyrhizobium japonicumand Identification of an Active Site Residue that Modulates Proline Reduction of the Flavin Adenine Dinucleotide Cofactor

Krishnan

Becker

2005

Biochemistry

View full text Add to dashboard Cite

PutA is a bifunctional flavoenzyme in bacteria that catalyzes the four-electron oxidation of proline to glutamate. In certain prokaryotes such as Escherichia coli, PutA is also a transcriptional repressor of the proline utilization (put) genes and thus is trifunctional. In this work, we have begun to assess differences between bifunctional and trifunctional PutA enzymes by examining the PutA protein from Bradyrhizobium japonicum (BjPutA). Primary structure analysis of BjPutA shows it lacks the DNA-binding domain of E. coli PutA (EcPutA). Consistent with this prediction, purified BjPutA does not exhibit DNA-binding activity in native gel mobility shift assays with promoter regions of the putA gene from B. japonicum. The catalytic and redox properties of BjPutA were characterized and a reduction potential (E m ) value of −0.132 V (pH 7.5) was determined for the bound FAD/ FADH 2 couple in BjPutA that is significantly more negative (∼ 55 mV) than the E m for EcPutAbound FAD. The more negative E m value thermodynamically limits proline reduction of the FAD cofactor in BjPutA. In the presence of phospholipids, reduction of BjPutA is stimulated suggesting lipids influence the FAD redox environment. Accordingly, an E m value of −-0.114 V (pH 7.5) was determined for BjPutA-bound FAD in the presence of polar lipids. The molecular basis for the lower reduction potential of FAD in BjPutA relative to EcPutA was explored by site-directed mutagenesis. Amino acid sequence alignment between BjPutA and EcPutA indicates only one difference in active site residues near the isoalloxazine ring of FAD: Val-402 in EcPutA is substituted at the analogous position in BjPutA with Ala-310. Replacement of A310 by Val in the BjPutA mutant A310V raised the reduction potential of bound FAD relative to wild-type BjPutA to an E m value of −0.09 V (pH 7.5). The > 40-mV positive shift in the potential of the BjPutA mutant A310V suggests that the corresponding Val residue in EcPutA helps poise the FAD redox potential for thermodynamically favored proline reduction thereby allowing EcPutA to be efficiently regulated by proline availability. Limited proteolysis of BjPutA under reducing conditions shows FAD reduction does not influence BjPutA conformation indicating further that the redox dependent regulation observed with EcPutA may be limited to trifunctional PutA homologues.Proline utilization is catalyzed by the flavoenzyme PutA (proline utilization A) in several microorganisms such as enteric bacteria Escherichia coli and Salmonella typhimurium and soil † This research was supported in part by NSF MCB0340912 and NIH GM61068, University of Nebraska Biochemistry Department and Redox Biology Center, and the Nebraska Agricultural Research Division, Journal Series No. 14857. This publication was also made possible by NIH Grant Number P20 RR-017675-02 from the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.Address correspondence to:...

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of a Bifunctional PutA Homologue fromBradyrhizobium japonicumand Identification of an Active Site Residue that Modulates Proline Reduction of the Flavin Adenine Dinucleotide Cofactor

Krishnan

Becker

2005

Biochemistry

View full text Add to dashboard Cite

show abstract

“…In the dehydrogenation of proline to P5C, two electrons are transferred from proline to a non-covalently bound FAD. Electrons from reduced FAD are then transferred to an acceptor in the electron transport chain to complete the catalytic cycle (3). Next, P5C, the product of the first reaction, is hydrolyzed to glutamate-␥-semialdehyde followed by subsequent transfer of two electrons to an NAD ϩ cofactor by the P5C dehydrogenase domain to yield glutamate.…”

mentioning

confidence: 99%

Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

Zhou

Kallhoff

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The PutA flavoprotein from Escherichia coli is a transcriptional repressor and a bifunctional enzyme that regulates and catalyzes proline oxidation. PutA represses transcription of genes putA and putP by binding to the control DNA region of the put regulon. The objective of this study is to define and characterize the DNA binding domain of PutA. The DNA binding activity of PutA, a 1320 amino acid polypeptide, has been localized to N-terminal residues 1-261. After exploring a potential DNA-binding region and an N-terminal deletion mutant of PutA, residues 1-90 (PutA90) were determined to contain DNA binding activity and stabilize the dimeric structure of PutA. Cell-based transcriptional assays demonstrate that PutA90 functions as a transcriptional repressor in vivo. The dissociation constant of PutA90 with the put control DNA was estimated to be 110 nM, which is slightly higher than that of the PutA-DNA complex (K d ϳ 45 nM). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbonhelix-helix DNA binding motif in residues 1-47. To test this prediction, we purified and characterized PutA47. PutA47 is shown to purify as an apparent dimer, to exhibit in vivo transcriptional activity, and to bind specifically to the put control DNA. In gel-mobility shift assays, PutA47 was observed to bind cooperatively to the put control DNA with an overall dissociation constant of 15 nM for the PutA47-DNA complex. Thus, Nterminal residues 1-47 are critical for DNA-binding and the dimeric structure of PutA. These results are consistent with the ribbon-helix-helix family of transcription factors.

show abstract

“…Figure 6A shows the enzymatic functions of EcPutA which are two-fold, involving a proline dehydrogenase (PRODH) domain that catalyzes the flavin-dependent oxidation of proline to D 1 -pyrroline-5-carboxylate (P5C) and a P5C dehydrogenase (P5CDH) domain that catalyzes the NAD þ -dependent conversion of g-glutamic acid semialdehyde (GSA) to glutamate (2,47,60). GSA is formed by the hydrolysis of P5C, a reaction which is presumably nonenzymatic.…”

Section: Puta Protein (Transcriptional Regulator and Membrane-bound Ementioning

confidence: 99%

Flavin Redox Switching of Protein Functions

Becker

Zhu

Moxley

2011

Antioxidants & Redox Signaling

View full text Add to dashboard Cite

Flavin cofactors impart remarkable catalytic diversity to enzymes, enabling them to participate in a broad array of biological processes. The properties of flavins also provide proteins with a versatile redox sensor that can be utilized for converting physiological signals such as cellular metabolism, light, and redox status into a unique functional output. The control of protein functions by the flavin redox state is important for transcriptional regulation, cell signaling pathways, and environmental adaptation. A significant number of proteins that have flavin redox switches are found in the Per-Arnt-Sim (PAS) domain family and include flavoproteins that act as photosensors and respond to changes in cellular redox conditions. Biochemical and structural studies of PAS domain flavoproteins have revealed key insights into how flavin redox changes are propagated to the surface of the protein and translated into a new functional output such as the binding of a target protein in a signaling pathway. Mechanistic details of proteins unrelated to the PAS domain are also emerging and provide novel examples of how the flavin redox state governs protein-membrane interactions in response to appropriate stimuli. Analysis of different flavin switch proteins reveals shared mechanistic themes for the regulation of protein structure and function by flavins.

show abstract

Proline Dehydrogenase from Escherichia coli K12

Cited by 68 publications

References 21 publications

Characterization of a Bifunctional PutA Homologue fromBradyrhizobium japonicumand Identification of an Active Site Residue that Modulates Proline Reduction of the Flavin Adenine Dinucleotide Cofactor

Characterization of a Bifunctional PutA Homologue fromBradyrhizobium japonicumand Identification of an Active Site Residue that Modulates Proline Reduction of the Flavin Adenine Dinucleotide Cofactor

Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

Flavin Redox Switching of Protein Functions

Contact Info

Product

Resources

About