Oxygen reactivity of PutA from Helicobacter species and proline-linked oxidative stress.
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 delta1-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 (PutA(Hh)) and Helicobacter pylori (PutA(Hp)) to provide new insights into proline metabolism in these gastrointestinal pathogens. Both PutA(Hh) and PutA(Hp) lack DNA binding activity, in contrast to PutA from Escherichia coli (PutA(Ec)), which both regulates and catalyzes proline utilization. PutA(Hh) and PutA(Hp) display catalytic activities similar to that of PutA(Ec) but have higher oxygen reactivity. PutA(Hh) and PutA(Hp) exhibit 100-fold-higher turnover numbers (approximately 30 min(-1)) than PutA(Ec) (<0. 3 min(-1)) using oxygen as an electron acceptor during catalytic turnover with proline. Consistent with increased oxygen reactivity, PutA(Hh) forms a reversible FAD-sulfite adduct. The significance of increased oxygen reactivity in PutA(Hh) and PutA(Hp) was probed by oxidative stress studies in E. coli. Expression of PutA(Ec) and PutA from Bradyrhizobium japonicum, which exhibit low oxygen reactivity, does not diminish stress survival rates of E. coli cell cultures. In contrast, PutA(Hp) and PutA(Hh) 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.
Krishnan
Navasona
N
Department of Biochemistry, University of Nebraska, N258 Beadle Center, Lincoln, Nebraska 68588, USA.
Becker
Donald F
DF
D001426:Bacterial Proteins / Q000378:metabolism*; D016998:Helicobacter / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D011394:Proline Oxidase / Q000378:metabolism*; D013045:Species Specificity
D001426:Bacterial Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria; D011392:Proline; D006861:Hydrogen Peroxide; D011394:Proline Oxidase; D010100:Oxygen