Back to Search
Start Over
Reverse protein engineering of a novel 4-domain copper nitrite reductase reveals functional regulation by protein-protein interaction.
- Source :
-
The FEBS journal [FEBS J] 2021 Jan; Vol. 288 (1), pp. 262-280. Date of Electronic Publication: 2020 Apr 28. - Publication Year :
- 2021
-
Abstract
- Cu-containing nitrite reductases that convert NO <subscript>2</subscript> <superscript>-</superscript> to NO are critical enzymes in nitrogen-based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4-domain CuNiR that has both cytochrome and cupredoxin domains fused at the N terminus and the other, a classical 2-domain CuNiR (Br <superscript>2D</superscript> NiR). We report the first enzymatic studies of a novel 4-domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3- and 2-domain variants, and Br <superscript>2D</superscript> NiR revealing up to ~ 500-fold difference in catalytic efficiency in comparison with classical 2-domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self-contained donor-acceptor system, we demonstrate that 4-domain BrNiR utilizes N-terminal tethering for downregulating enzymatic activity instead. Both Br <superscript>2D</superscript> NiR and an engineered 2-domain variant of BrNiR (Δ(Cytc-Cup) BrNiR) have 3 to 5% NiR activity compared to the well-characterized 2-domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc-Cup) BrNiR and Br <superscript>2D</superscript> NiR with classical 2-domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally occurring 4-domain CuNiR and its engineered 3- and 2-domain variants. The reverse protein engineering approach utilized here has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes. ENZYME: Copper-containing nitrite reductase (CuNiR) (EC 1.7.2.1). DATABASE: The atomic coordinate and structure factor of Δ(Cytc-Cup) BrNiR and Br <superscript>2D</superscript> NiR have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code 6THE and 6THF, respectively.<br /> (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Subjects :
- Achromobacter cycloclastes enzymology
Achromobacter cycloclastes genetics
Alcaligenes enzymology
Alcaligenes genetics
Amino Acid Sequence
Azurin chemistry
Azurin genetics
Azurin metabolism
Bacterial Proteins genetics
Bacterial Proteins metabolism
Bradyrhizobium enzymology
Bradyrhizobium genetics
Catalytic Domain
Cloning, Molecular
Copper metabolism
Crystallography, X-Ray
Cytochromes c chemistry
Cytochromes c genetics
Cytochromes c metabolism
Electrons
Escherichia coli genetics
Escherichia coli metabolism
Gene Expression
Genetic Vectors chemistry
Genetic Vectors metabolism
Models, Molecular
Nitrite Reductases genetics
Nitrite Reductases metabolism
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Engineering methods
Protein Interaction Domains and Motifs
Protons
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Reverse Genetics methods
Sequence Alignment
Sequence Homology, Amino Acid
Substrate Specificity
Achromobacter cycloclastes chemistry
Alcaligenes chemistry
Bacterial Proteins chemistry
Bradyrhizobium chemistry
Copper chemistry
Nitrite Reductases chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1742-4658
- Volume :
- 288
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- The FEBS journal
- Publication Type :
- Academic Journal
- Accession number :
- 32255260
- Full Text :
- https://doi.org/10.1111/febs.15324