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Journal of Bacteriology, February 2006, p. 1227-1235, Vol. 188, No. 4
0021-9193/06/$08.00+0     doi:10.1128/JB.188.4.1227-1235.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Oxygen Reactivity of PutA from Helicobacter Species and Proline-Linked Oxidative Stress

Navasona Krishnan and Donald F. Becker*

Department of Biochemistry, Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588

Received 27 September 2005/ Accepted 11 November 2005

Proline is converted to glutamate in two successive steps by the proline utilization A (PutA) flavoenzyme in gram-negative bacteria. PutA contains a proline dehydrogenase domain that catalyzes the flavin adenine dinucleotide (FAD)-dependent oxidation of proline to {Delta}1-pyrroline-5-carboxylate (P5C) and a P5C dehydrogenase domain that catalyzes the NAD+-dependent oxidation of P5C to glutamate. Here, we characterize PutA from Helicobacter hepaticus (PutAHh) and Helicobacter pylori (PutAHp) to provide new insights into proline metabolism in these gastrointestinal pathogens. Both PutAHh and PutAHp lack DNA binding activity, in contrast to PutA from Escherichia coli (PutAEc), which both regulates and catalyzes proline utilization. PutAHh and PutAHp display catalytic activities similar to that of PutAEc but have higher oxygen reactivity. PutAHh and PutAHp exhibit 100-fold-higher turnover numbers (~30 min–1) than PutAEc (<0. 3 min–1) using oxygen as an electron acceptor during catalytic turnover with proline. Consistent with increased oxygen reactivity, PutAHh forms a reversible FAD-sulfite adduct. The significance of increased oxygen reactivity in PutAHh and PutAHp was probed by oxidative stress studies in E. coli. Expression of PutAEc and PutA from Bradyrhizobium japonicum, which exhibit low oxygen reactivity, does not diminish stress survival rates of E. coli cell cultures. In contrast, PutAHp and PutAHh expression dramatically reduces E. coli cell survival and is correlated with relatively lower proline levels and increased hydrogen peroxide formation. The discovery of reduced oxygen species formation by PutA suggests that proline catabolism may influence redox homeostasis in the ecological niches of these Helicobacter species.

* Corresponding author. Mailing address: Department of Biochemistry, University of Nebraska, N258 Beadle Center, Lincoln, NE 68588. Phone: (402) 472-9652. Fax: (402) 472-7842. E-mail: dbecker3{at}unl.edu.

Journal of Bacteriology, February 2006, p. 1227-1235, Vol. 188, No. 4
0021-9193/06/$08.00+0     doi:10.1128/JB.188.4.1227-1235.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Fernandez-Pinar, R., Ramos, J. L., Rodriguez-Herva, J. J., Espinosa-Urgel, M. (2008). A Two-Component Regulatory System Integrates Redox State and Population Density Sensing in Pseudomonas putida. J. Bacteriol. 190: 7666-7674 [Abstract] [Full Text]  
  • Krishnan, N., Doster, A. R., Duhamel, G. E., Becker, D. F. (2008). Characterization of a Helicobacter hepaticus putA Mutant Strain in Host Colonization and Oxidative Stress. Infect. Immun. 76: 3037-3044 [Abstract] [Full Text]  
  • White, T. A., Krishnan, N., Becker, D. F., Tanner, J. J. (2007). Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus. J. Biol. Chem. 282: 14316-14327 [Abstract] [Full Text]  


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