Oxygen reactivity of PutA from Helicobacter species and proline-linked oxidative stress

Abstract

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.

FIG. 1.

Proline reduction of PutA_Hh. PutA_Hh was titrated with proline under anaerobic conditions in 50 mM potassium phosphate (pH 7.5) at 25°C. Spectra were recorded 10 min after the addition of proline. Curves 1 to 9, 0, 0.02, 0.04, 0.05, 0.07, 0.37, 0.97, 1.97, and 2.97 mM proline, respectively.

FIG. 2.

Reaction of PutA_Hh with sodium sulfite. (Top) PutA_Hh was incubated with different concentrations of sodium sulfite in 50 mM potassium phosphate buffer (pH 7.5) at 25°C for 15 min. Curves 1 to 6, 0, 0.05, 0.1, 0.5, 1, and 4 mM sodium sulfite, respectively. Inset is a plot of the decrease in absorbance (ΔAbs) at 451 nm versus sulfite concentration. The data are fit to a single-binding-site isotherm with a dissociation constant value of 0.4 ± 0.06 mM sulfite. (Bottom) Spectral changes that occur upon the addition of l-THFA to the sulfite-PutA_Hh mixture. Curves 1 to 3, 0, 1, and 5 mM l-THFA, respectively.

FIG. 3.

Acute stress treatment of E. coli cell cultures expressing the various PutA enzymes. E. coli cells were incubated with 5 mM H₂O₂, 5 mM proline, or a mixture of 5 mM each of proline and l-THFA in M9 minimal medium for 1 h at 37°C. After the incubation, cells were serially diluted and plated onto LB agar plates containing ampicillin and grown at 37°C. Images were recorded after 16 h of growth. The control cells contain the pET14b vector alone.

FIG. 4.

Cell survival rates of E. coli cells expressing different PutA enzymes. E. coli cells were incubated with 5 mM H₂O₂ (black), 5 mM proline (gray), or a mixture of 5 mM proline and 5 mM l-THFA (diagonal stripes) in M9 minimal medium for 1 h at 37°C. After incubation, cells were serially diluted and plated onto LB agar plates and grown at 37°C for 16 h. Colonies were then counted to estimate the percent survival rates, which are the average values from at least six independent experiments. Percent survival was calculated as follows: [(number of colonies after addition of reagent)/(number of colonies with no addition of reagent)] × 100. EcPutA, PutA_Ec; BjPutA, PutA_Bj; HhPutA, PutA_Hh; HpPutA, PutA_Hp.

FIG. 5.

Proline levels in E. coli cell suspensions expressing different PutA enzymes. E. coli cells were incubated and supplemented with no proline (black), 5 mM proline (gray), or a mixture of 5 mM proline and 5 mM l-THFA (diagonal lines) in M9 minimal medium for 1 h at 37°C. Proline levels were determined from lysed cell supernatants and are reported as estimates of intracellular concentrations. The values are an average of three independent experiments. EcPutA, PutA_Ec; BjPutA, PutA_Bj; HhPutA, PutA_Hh; HpPutA, PutA_Hp.

FIG. 6.

Time course of H₂O₂ released from E. coli cells harboring different PutA enzymes. Extracellular H₂O₂ was measured in E. coli cell cultures containing PutA_Ec (▪), PutA_Bj (⧫), PutA_Hh (•), and PutA_Hp (▴) in M9 minimal medium. Cultures were supplemented with no proline (A), 5 mM proline (B), or a mixture of 5 mM proline and 5 mM l-THFA (C). The values are an average of three independent experiments.