TY - JOUR
T1 - Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass
AU - Campeciño, Julius
AU - Lagishetty, Satyanarayana
AU - Wawrzak, Zdzislaw
AU - Alfaro, Victor Sosa
AU - Lehnert, Nicolai
AU - Reguera, Gemma
AU - Hu, Jian
AU - Hegg, Eric L.
N1 - Funding Information:
Funding and additional information—This work was supported by the U.S. Department of Energy, Office of Science, Basic Energy Sciences, under award no. DE-SC0017952 (to G. R. and E. L. H.) and award no. DE-SC0018173 (to N. L.).
Publisher Copyright:
© 2020 Campeciño et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2020/8/14
Y1 - 2020/8/14
N2 - 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 Ca21-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 Ca21-dependent enzymes, suggesting convergent evolution.
AB - 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 Ca21-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 Ca21-dependent enzymes, suggesting convergent evolution.
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U2 - 10.1074/jbc.ra120.013981
DO - 10.1074/jbc.ra120.013981
M3 - Article
C2 - 32518164
AN - SCOPUS:85089786955
VL - 295
SP - 11455
EP - 11465
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 33
ER -