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 Δ¹-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 (~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.