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 Scopus citations

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

Keywords

  • Proline catabolism
  • Substrate channeling

ASJC Scopus subject areas

  • General

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    Srivastava, D., Schuermann, J. P., White, T. A., Krishnan, N., Sanyal, N., Hurad, G. L., Tan, A., Henzl, M. T., Becker, D. F., & Tanner, J. J. (2010). Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum. Proceedings of the National Academy of Sciences of the United States of America, 107(7), 2878-2883. https://doi.org/10.1073/pnas.0906101107