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Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass.

ABSTRACT: Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca²⁺-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca²⁺-dependent enzymes, suggesting convergent evolution.

SUBMITTER: Campecino J

PROVIDER: S-EPMC7450111 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass.

Campeciño Julius J Lagishetty Satyanarayana S Wawrzak Zdzislaw Z Sosa Alfaro Victor V Lehnert Nicolai N Reguera Gemma G Hu Jian J Hegg Eric L EL

The Journal of biological chemistry 20200609 33

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and cha ...[more]

PMID: 32518164

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Project description:The versatile soil bacterium Anaeromyxobacter dehalogenans lacks the hallmark denitrification genes nirS and nirK (encoding NO2-→NO reductases) and couples growth to NO3- reduction to NH4+ (respiratory ammonification) and to N2O reduction to N2A. dehalogenans also grows by reducing Fe(III) to Fe(II), which chemically reacts with NO2- to form N2O (i.e., chemodenitrification). Following the addition of 100 μmol of NO3- or NO2- to Fe(III)-grown axenic cultures of A. dehalogenans, 54 (±7) μmol and 113 (±2) μmol N2O-N, respectively, were produced and subsequently consumed. The conversion of NO3- to N2 in the presence of Fe(II) through linked biotic-abiotic reactions represents an unrecognized ecophysiology of A. dehalogenans The new findings demonstrate that the assessment of gene content alone is insufficient to predict microbial denitrification potential and N loss (i.e., the formation of gaseous N products). A survey of complete bacterial genomes in the NCBI Reference Sequence database coupled with available physiological information revealed that organisms lacking nirS or nirK but with Fe(III) reduction potential and genes for NO3- and N2O reduction are not rare, indicating that NO3- reduction to N2 through linked biotic-abiotic reactions is not limited to A. dehalogenans Considering the ubiquity of iron in soils and sediments and the broad distribution of dissimilatory Fe(III) and NO3- reducers, denitrification independent of NO-forming NO2- reductases (through combined biotic-abiotic reactions) may have substantial contributions to N loss and N2O flux.IMPORTANCE Current attempts to gauge N loss from soils rely on the quantitative measurement of nirK and nirS genes and/or transcripts. In the presence of iron, the common soil bacterium Anaeromyxobacter dehalogenans is capable of denitrification and the production of N2 without the key denitrification genes nirK and nirS Such chemodenitrifiers denitrify through combined biotic and abiotic reactions and have potentially large contributions to N loss to the atmosphere and fill a heretofore unrecognized ecological niche in soil ecosystems. The findings emphasize that the comprehensive understanding of N flux and the accurate assessment of denitrification potential can be achieved only when integrated studies of interlinked biogeochemical cycles are performed.

Dataset Information

Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass.

Publications

Cytochrome <i>c</i> nitrite reductase from the bacterium <i>Geobacter lovleyi</i> represents a new NrfA subclass.

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