Reduction of a Heme Cofactor Initiates N-Nitroglycine Degradation by NnlA

Abstract

Linear nitramines are potentially carcinogenic environmental contaminants. The NnlA enzyme from Variovorax sp. strain JS1663 degrades the nitramine N-nitroglycine (NNG)-a natural product produced by some bacteria-to glyoxylate and nitrite (NO₂^-). Ammonium (NH₄⁺) was predicted as the third product of this reaction. A source of nonheme Fe^II was shown to be required for initiation of NnlA activity. However, the role of this Fe^II for NnlA activity was unclear. This study reveals that NnlA contains a b-type heme cofactor. Reduction of this heme-either by a nonheme iron source or dithionite-is required to initiate NnlA activity. Therefore, Fe^II is not an essential substrate for holoenzyme activity. Our data show that reduced NnlA (Fe^II-NnlA) catalyzes at least 100 turnovers and does not require O₂. Finally, NH₄⁺ was verified as the third product, accounting for the complete nitrogen mass balance. Size exclusion chromatography showed that NnlA is a dimer in solution. Additionally, Fe^II-NnlA is oxidized by O₂ and NO₂^- and stably binds carbon monoxide (CO) and nitric oxide (NO). These are characteristics shared with heme-binding PAS domains. Furthermore, a structural homology model of NnlA was generated using the PAS domain from Pseudomonas aeruginosa Aer2 as a template. The structural homology model suggested His⁷³ is the axial ligand of the NnlA heme. Site-directed mutagenesis of His⁷³ to alanine decreased the heme occupancy of NnlA and eliminated NNG activity, validating the homology model. We conclude that NnlA forms a homodimeric heme-binding PAS domain protein that requires reduction for initiation of the activity. IMPORTANCE Linear nitramines are potential carcinogens. These compounds result from environmental degradation of high-energy cyclic nitramines and as by-products of carbon capture technologies. Mechanistic understanding of the biodegradation of these compounds is critical to inform strategies for their remediation. Biodegradation of NNG by NnlA from Variovorax sp. strain JS 1663 requires nonheme iron, but its role is unclear. This study shows that nonheme iron is unnecessary. Instead, our study reveals that NnlA contains a heme cofactor, the reduction of which is critical for activating NNG degradation activity. These studies constrain the proposals for NnlA reaction mechanisms, thereby informing mechanistic studies of degradation of anthropogenic nitramine contaminants. In addition, these results will inform future work to design biocatalysts to degrade these nitramine contaminants.

Keywords: N-nitroglycine; PAS domain; biodegradation; enzymology; heme; nitramine; nitrogen; nitrogen metabolism.

FIG 1

Product distributions of nitramine degrading enzymes, including (A) aerobic degradation of RDX by XplA, and (B) degradation of NNG by NnlA. Reactants and products not yet verified are shown in square brackets.

FIG 2

UV-visible absorption spectra of as isolated NnlA expressed in the absence (red trace) or presence (black trace) of 5-ALA. Both spectra were measured in 50 mM phosphate buffer at pH 7.2.

FIG 3

Treatment of Fe^II-NnlA with (A) air, (B) NO, or (C) CO. Conditions before initiation of reaction by inversion of sample into gas headspace were 10 μM Fe^II-NnlA in either in 100 mM Tris-HCl pH 7.6 (panel A) or 100 mM tricine buffer at pH 8 (panels B and C).

FIG 4

Overlaid representative LC-MS EICs monitoring molecular anions of NNG (m/z 119.0) and glyoxylate (m/z 73.0) in samples containing 500 μM NNG and 10 μM dithionite (dithionite/NNG), 10 μM as isolated NnlA (Fe^III-NnlA), or 10 μM reduced NnlA (Fe^II-NnlA). Samples were incubated for 30 min at room temperature in deoxygenated 20 mM phosphate buffer, pH 7.2. The Fe^II-NnlA/O₂ sample was incubated in air-saturated buffer. Dashed and dotted gray lines indicate elution time of glyoxylate and NNG in standard solutions.

FIG 5

UV-visible spectra of reduced NnlA treated with either NNG (panel A) or NO₂^– (panel B) under anaerobic conditions. Reaction conditions: (panel A) 3 μM Fe^II-NnlA, 133 μM NNG, in deoxygenated 20 mM phosphate, pH 7.5 incubated for 5 h at room temperature; (panel B) 3 μM Fe^II-NnlA with 133 μM NO₂^– in deoxygenated 20 mM phosphate, pH 7.5 at room temperature.

FIG 6

Structural homology model of NnlA overlaid on the crystal structure of the CN^– complexed PAS domain from Pa Aer2 (PDB: 3VOL; gray). Peptide backbone of NnlA structural homology model shown as green cartoon and Pa Aer2 backbone shown as gray cartoon. Iron atom shown as orange sphere, oxygen and nitrogen atoms shown in red and blue, respectively. Structural homology model was generated by SWISS-MODEL.

FIG 7

UV-visible absorption spectra of 20 μM wild-type or 17 μM H73A NnlA. All samples prepared in 100 mM tricine buffer, pH 8.

FIG 8

Summary of NnlA activity.

FIG 9

Proposed roles of heme cofactor in NnlA.