PT - JOURNAL ARTICLE AU - Kara A. Strickland AU - Ashley A. Holland AU - Alan Trudeau AU - Ilana Szlamkowicz AU - Melanie J. Beazley AU - Vasileios A. Anagnostopoulos AU - David E. Graham AU - Jonathan D. Caranto TI - Reduction of a heme cofactor initiates N-nitroglycine degradation by NnlA AID - 10.1101/2022.06.19.496723 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.06.19.496723 4099 - http://biorxiv.org/content/early/2022/06/19/2022.06.19.496723.short 4100 - http://biorxiv.org/content/early/2022/06/19/2022.06.19.496723.full AB - The NnlA enzyme from Variovorax sp. strain JS1663 degrades the linear 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 non-heme FeII was shown to be required for initiation of NnlA activity. However, it was unclear if this FeII was being used as a metallocofactor or a reductant. This study reveals that NnlA contains a b-type heme cofactor. Reduction of this heme is required to initiate NnlA activity. Reduction can occur either by addition of a non-heme FeII source or by reduction with dithionite. Therefore, FeII is not an essential substrate for holoenzyme activity. Data are presented showing that reduced NnlA (FeII-NnlA) can catalyze at least 100 turnovers. In addition, this catalysis occurred in the absence of 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, FeII-NnlA is oxidized by O2 and NO2− and binds carbon monoxide (CO) and nitric oxide (NO). These are characteristics shared with PAS domains; NnlA was previously shown to exhibit homology with such domains. Providing further evidence, a structural homology model of NnlA was generated based on the structure of the PAS domain from Pseudomonas aeruginosa Aer2. 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, providing evidence that the homology model is valid. 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 linear nitramines is critical to inform approaches for their remediation. The best understood biodegradation of a linear nitramine is NNG degradation by NnlA from Variovorax sp. strain JS 1663; however, it is unclear why non-heme iron was required to initiate enzymatic turnover. This study shows that non-heme 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 future work to design biocatalysts to degrade these nitramine contaminants.Competing Interest StatementThe authors have declared no competing interest.