Involvement of the β3-α3 Loop of the Proline Dehydrogenase Domain in Allosteric Regulation of Membrane Association of Proline Utilization A

Zhu, Weidong; Haile, Ashley M.; Singh, Ranjan K.; Larson, J.D.; Smithen, Danielle; Chan, Jie Ying; Tanner, John J.; Becker, Donald F.

doi:10.1021/bi400396g

Cited by 17 publications

(52 citation statements)

References 28 publications

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“…To measure the effect of proline oxidation on katG expression, AL441 cells were grown in glucose minimal medium to an OD 600 of 0.3 before treatment with 10 mM L-proline and L-THFA. Samples were collected at designated time points, and ␤-galactosidase activities were measured as described previously (30). To determine the effect of H 2 O 2 on katG expression, cells were cultured as described above and then treated with different concentrations of H 2 O 2 for 30 min, followed by measurement of ␤-galactosidase activity.…”

Section: Methodsmentioning

confidence: 99%

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Proline Metabolism IncreaseskatGExpression and Oxidative Stress Resistance in Escherichia coli

2014

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The oxidation of L-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and ⌬ 1 -pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ⌬katG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.T he conversion of L-proline to glutamate is a four-electron oxidation process that is coordinated in two successive steps by the enzymes proline dehydrogenase (PRODH) and ⌬ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH) (Fig. 1) (1). In eukaryotes, PRODH and P5CDH are separately encoded enzymes localized in the mitochondrion. In Gram-negative bacteria, PRODH and P5CDH are combined into a bifunctional enzyme known as proline utilization A (PutA) (1, 2). The PRODH domain contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor and couples the two-electron oxidation of proline to the reduction of ubiquinone in the cytoplasmic membrane (3). The product of the PRODH reaction, ⌬ 1 -pyrroline-5-carboxylate (P5C), is subsequently hydrolyzed to glutamate-␥-semialdehyde (GSA), which is then oxidized to glutamate by the NAD ϩ -dependent P5CDH domain (2). In certain Gram-negative bacteria such as Escherichia coli, PutA also has an N-terminal ribbon-helix-helix (RHH) DNA-binding domain (residues 1 to 47) (4). The RHH domain enables PutA to act as an autogenous transcriptional regulator of the putA and putP (highaffinity Na ϩ -proline transporter) genes (4). PutA represses put gene expression by binding to five operator sites in the put regulatory region (5). Transcription of the put genes is activated by proline, which causes a r...

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Section: Methodsmentioning

confidence: 99%

“…To determine the effect of H 2 O 2 on katG expression, cells were cultured as described above and then treated with different concentrations of H 2 O 2 for 30 min, followed by measurement of ␤-galactosidase activity. ␤-Galactosidase activity assays were performed as previously described (30) and are reported in Miller units (29).…”

Section: Methodsmentioning

confidence: 99%

Proline Metabolism IncreaseskatGExpression and Oxidative Stress Resistance in Escherichia coli

2014

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“…During periods of low proline availability, PutA represses the put regulon by binding to putA/P promoter regions (9). When high concentrations of proline are present, PutA repression is relieved by proline reduction of the FAD cofactor, which induces PutA binding to the inner cytoplasmic membrane, a process known as redox functional switching (8,10,11).…”

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confidence: 99%

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

Moxley

Sanyal

Krishnan

et al. 2014

Journal of Biological Chemistry

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Background:PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism. Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step. Conclusion: Substrate channeling in PutA exhibits hysteretic behavior. Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation.

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“…The mechanism whereby PutA switches from a DNA-binding protein to a membrane-bound flavoenzyme is mediated by a proline-induced conformational change that is hypothesized to initiate at the flavin and traverse through the surrounding protein [85]. A proline-dependent conformational change is supported in part by limited proteolysis data that show praline reduction of the flavin induces a global conformational change involving the α-domain of EcPutA [48].…”

Section: Puta Is a Flavin Switch Proteinmentioning

confidence: 99%

“…For example, site-directed mutagenesis implicated the linkage between the FAD N(5) and Glu372 as being critical for functional switching of EcPutA [66, 85]. Mutation of Arg431 or substitution of normal FAD with 5-deazaFAD, which lacks N(5), blocks EcPutA membrane binding [66].…”

Section: Puta Is a Flavin Switch Proteinmentioning

confidence: 99%

Structure, function, and mechanism of proline utilization A (PutA)

Liu

Becker

Tanner

2017

Archives of Biochemistry and Biophysics

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Proline has important roles in multiple biological processes such as cellular bioenergetics, cell growth, oxidative and osmotic stress response, protein folding and stability, and redox signaling. The proline catabolic pathway, which forms glutamate, enables organisms to utilize proline as a carbon, nitrogen, and energy source. FAD-dependent proline dehydrogenase (PRODH) and NAD+-dependent glutamate semialdehyde dehydrogenase (GSALDH) convert proline to glutamate in two sequential oxidative steps. Depletion of PRODH and GSALDH in humans leads to hyperprolinemia, which is associated with mental disorders such as schizophrenia. Also, some pathogens require proline catabolism for virulence. A unique aspect of proline catabolism is the multifunctional proline utilization A (PutA) enzyme found in Gram-negative bacteria. PutA is a large (> 1000 residues) bifunctional enzyme that combines PRODH and GSALDH activities into one polypeptide chain. In addition, some PutAs function as a DNA-binding transcriptional repressor of proline utilization genes. This review describes several attributes of PutA that make it a remarkable flavoenzyme: (1) diversity of oligomeric state and quaternary structure; (2) substrate channeling and enzyme hysteresis; (3) DNA-binding activity and transcriptional repressor function; and (4) flavin redox dependent changes in subcellular location and function in response to proline (functional switching).

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Involvement of the β3-α3 Loop of the Proline Dehydrogenase Domain in Allosteric Regulation of Membrane Association of Proline Utilization A

Cited by 17 publications

References 28 publications

Proline Metabolism IncreaseskatGExpression and Oxidative Stress Resistance in Escherichia coli

Proline Metabolism IncreaseskatGExpression and Oxidative Stress Resistance in Escherichia coli

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

Structure, function, and mechanism of proline utilization A (PutA)

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