Small-angle X-ray Scattering Studies of the Oligomeric State and Quaternary Structure of the Trifunctional Proline Utilization A (PutA) Flavoprotein from Escherichia coli

Singh, Ranjan K.; Larson, J.D.; Zhu, Weidong; Rambo, Robert P.; Hura, Greg L.; Becker, Donald F.; Tanner, John J.

doi:10.1074/jbc.m111.292474

Cited by 17 publications

(39 citation statements)

References 51 publications

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“…The poor prediction of the oligomeric state for Escherichia coli proline utilization A is likely to be owing to the unusual shape of this protein. SAXS shape reconstructions show that the dimeric particle is highly elongated, with dimensions of 205 Â 85 Â 55 Å (Singh et al, 2011). As shown next, highly elongated proteins deviate substantially from power-law behavior.…”

Section: Predicting Oligomeric State From An Experimental Measurementmentioning

confidence: 87%

Empirical power laws for the radii of gyration of protein oligomers

Tanner

2016

Acta Crystallogr D Struct Biol

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The radius of gyration is a fundamental structural parameter that is particularly useful for describing polymers. It has been known since Flory's seminal work in the mid-20th century that polymers show a power-law dependence, where the radius of gyration is proportional to the number of residues raised to a power. The power-law exponent has been measured experimentally for denatured proteins and derived empirically for folded monomeric proteins using crystal structures. Here, the biological assemblies in the Protein Data Bank are surveyed to derive the power-law parameters for protein oligomers having degrees of oligomerization of 2–6 and 8. The power-law exponents for oligomers span a narrow range of 0.38–0.41, which is close to the value of 0.40 obtained for monomers. This result shows that protein oligomers exhibit essentially the same power-law behavior as monomers. A simple power-law formula is provided for estimating the oligomeric state from an experimental measurement of the radius of gyration. Several proteins in the Protein Data Bank are found to deviate substantially from power-law behavior by having an atypically large radius of gyration. Some of the outliers have highly elongated structures, such as coiled coils. For coiled coils, the radius of gyration does not follow a power law and instead scales linearly with the number of residues in the oligomer. Other outliers are proteins whose oligomeric state or quaternary structure is incorrectly annotated in the Protein Data Bank. The power laws could be used to identify such errors and help prevent them in future depositions.

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Section: Predicting Oligomeric State From An Experimental Measurementmentioning

confidence: 87%

Empirical power laws for the radii of gyration of protein oligomers

Tanner

2016

Acta Crystallogr D Struct Biol

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“…Experiments that are described as anaerobic were subjected to vacuum/nitrogen gas cycles followed by the addition of protocatechuate dioxygenase (0.05 unit/ml) and protocatechuic acid (100 M) to scrub the remaining oxygen as described previously (17). (20), which is based on small angle x-ray scattering, crystal structures of EcPutA DNA-binding and PRODH domains, and homology to BjPutA. The DNA-binding, PRODH, and P5CDH domains are colored as in the domain diagram.…”

Section: Experimental Prodceduresmentioning

confidence: 99%

“…Tyr-58 is proposed to move and thereby allow ammonia to enter the 40 Å channeling pathway to the NAD ϩ synthetase active site (48). Molecular dynamics simulations of PutA could provide key insights to help explain our kinetic observations and further test a channel gating hypothesis (12,20). Other conformational changes not yet known may also contribute to the activation of EcPutA.…”

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confidence: 99%

“…Although an x-ray crystal structure of full-length EcPutA is not available, a model of EcPutA (Fig. 2B) was recently constructed using the crystal structures of the PRODH and DNA-binding domains, a homology model of the P5CDH domain, and small angle x-ray scattering data of full-length EcPutA (20). Interestingly, the small angle x-ray scattering data showed that EcPutA and BjPutA have completely different oligomeric states and quaternary structures, which results from the additional DNA-binding and C-terminal domains of EcPutA.…”

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confidence: 99%

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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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“…SAXS showed that EcPutA forms an elongated V-shaped dimer with radius of gyration ( R g ) of 63 Å and dimensions of 205 × 85 × 55 Å [42]. Domain deletion analysis and SAXS rigid body modeling show the RHH domain mediates dimerization at the centroid of the particle, and the catalytic modules (residues 85–1320) reside in the large outer lobes.…”

Section: Three-dimensional Structure Of Putamentioning

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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Small-angle X-ray Scattering Studies of the Oligomeric State and Quaternary Structure of the Trifunctional Proline Utilization A (PutA) Flavoprotein from Escherichia coli

Cited by 17 publications

References 51 publications

Empirical power laws for the radii of gyration of protein oligomers

Empirical power laws for the radii of gyration of protein oligomers

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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