Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

Dhiraj Srivastava, Jonathan P. Schuermann, Tommi A. White, Navasona Krishnan, Nikhilesh Sanyal, Greg L. Hurad, Anmin Tan, Michael T. Henzl, Donald F Becker, John J. Tanner

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD +-dependent Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 Å resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 Å. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 Å and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Δ1-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Δ1-pyrroline-5- carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.

Original languageEnglish (US)
Pages (from-to)2878-2883
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number7
DOIs
StatePublished - Feb 16 2010

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Bradyrhizobium
Proline
Glutamic Acid
Proline Oxidase
Oxidoreductases
1-Pyrroline-5-Carboxylate Dehydrogenase
Catalytic Domain
Flavin-Adenine Dinucleotide
Ultracentrifugation
Protein Subunits
NAD
Hydrolysis
X-Rays
delta-1-pyrroline-5-carboxylate

Keywords

  • Proline catabolism
  • Substrate channeling

ASJC Scopus subject areas

  • General

Cite this

Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum. / Srivastava, Dhiraj; Schuermann, Jonathan P.; White, Tommi A.; Krishnan, Navasona; Sanyal, Nikhilesh; Hurad, Greg L.; Tan, Anmin; Henzl, Michael T.; Becker, Donald F; Tanner, John J.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 7, 16.02.2010, p. 2878-2883.

Research output: Contribution to journalArticle

Srivastava, Dhiraj ; Schuermann, Jonathan P. ; White, Tommi A. ; Krishnan, Navasona ; Sanyal, Nikhilesh ; Hurad, Greg L. ; Tan, Anmin ; Henzl, Michael T. ; Becker, Donald F ; Tanner, John J. / Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum. In: Proceedings of the National Academy of Sciences of the United States of America. 2010 ; Vol. 107, No. 7. pp. 2878-2883.
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AU - Srivastava, Dhiraj

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AU - Sanyal, Nikhilesh

AU - Hurad, Greg L.

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N2 - The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD +-dependent Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 Å resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 Å. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 Å and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Δ1-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Δ1-pyrroline-5- carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.

AB - The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD +-dependent Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 Å resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 Å. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 Å and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Δ1-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Δ1-pyrroline-5- carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.

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