Primary Amine Oxidase of Escherichia coli Is a Metabolic Enzyme that Can Use a Human Leukocyte Molecule as a Substrate

Abstract

Escherichia coli amine oxidase (ECAO), encoded by the tynA gene, catalyzes the oxidative deamination of aromatic amines into aldehydes through a well-established mechanism, but its exact biological role is unknown. We investigated the role of ECAO by screening environmental and human isolates for tynA and characterizing a tynA-deletion strain using microarray analysis and biochemical studies. The presence of tynA did not correlate with pathogenicity. In tynA+ Escherichia coli strains, ECAO enabled bacterial growth in phenylethylamine, and the resultant H2O2 was released into the growth medium. Some aminoglycoside antibiotics inhibited the enzymatic activity of ECAO, which could affect the growth of tynA+ bacteria. Our results suggest that tynA is a reserve gene used under stringent environmental conditions in which ECAO may, due to its production of H2O2, provide a growth advantage over other bacteria that are unable to manage high levels of this oxidant. In addition, ECAO, which resembles the human homolog hAOC3, is able to process an unknown substrate on human leukocytes.

Fig 1. The time and medium dependence of ECAO expression and activity.

(A) The kinetics of ECAO induction in E. coli K-12 (wt) cultured in M9-lactose medium supplemented with 5 mM PEA (n = 4). The 0 h time point corresponds to the time when the cells were diluted into PEA-containing culture medium. (B) The activity of ECAO is induced in E. coli K-12 (wt), but not in our ΔtynA strain, by the presence of both lactose and PEA 6 h after diluting the bacterial cultures in M9-lactose-PEA media (n = 3). (C) The H₂O₂ production of wt K-12 E. coli and ΔtynA bacteria in stationary phase in M9-lactose-PEA medium (** p < 0.01). The means ± SEMs are shown.

Fig 2. Different ECAO inhibitors.

(A) The relative inhibition of ECAO by semicarbazide (SC) and (B) the hAOC3 inhibitor BTT-2052. (C) The effect of different aminoglycosides (1 mM) on the activity of ECAO (** p < 0.01). (D) The inhibition curves of different aminoglycosides on ECAO activity. (E) The pairwise comparison of inhibition curves between BTT-2052 and amikacin.

Fig 3. Pathways altered by tynA knockout.

(A) The phenylethylamine metabolic pathway is down-regulated (green) in the ΔtynA strain when compared to the wt strain in M9-lactose-PEA medium. (B) The oxidation of propionate to pyruvate is up-regulated (red) in the ΔtynA strain. Pathways are depicted according to Brock et al. and Zeng et al. [5,41].

Fig 4. ECAO is able to use human granulocytes as a substrate.

We detected the production of H₂O₂ by ECAO when granulocytes were the only source of substrate (Cells + ECAO), and the activity was inhibited by semicarbazide (Cells + ECAO + SC). The activity is significantly higher than without ECAO (Cells only, p = 0.0003; cells with SC, p = 0.0004; without cells, p = 0.017, and with added SC inhibitor [Cells+ECAO+SC], p = 0.004). The mean of three independent experiments is shown ± SEM. Statistical significance was calculated with paired t-test.