Reverse protein engineering of a novel 4-domain copper nitrite reductase reveals functional regulation by protein-protein interaction.

Publisher: Published by Blackwell Pub. on behalf of the Federation of European Biochemical Societies Country of Publication: England NLM ID: 101229646 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1742-4658 (Electronic) Linking ISSN: 1742464X NLM ISO Abbreviation: FEBS J Subsets: MEDLINE

Original Publication: Oxford, UK : Published by Blackwell Pub. on behalf of the Federation of European Biochemical Societies, c2005-

Achromobacter cycloclastes/*chemistry ; Alcaligenes/*chemistry ; Bacterial Proteins/*chemistry ; Bradyrhizobium/*chemistry ; Copper/*chemistry ; Nitrite Reductases/*chemistry; 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

Cu-containing nitrite reductases that convert NO 2^- 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 ^2D 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 ^2D 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 ^2D 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 ^2D 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 ^2D NiR have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code 6THE and 6THF, respectively.
(© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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BB/N013972/1 United Kingdom BB_ Biotechnology and Biological Sciences Research Council

Keywords: catalysis; denitrification; electron transfer; multidomain protein; protein engineering

0 (Bacterial Proteins)
0 (Protons)
0 (Recombinant Proteins)
0 (cupredoxin)
12284-43-4 (Azurin)
789U1901C5 (Copper)
9007-43-6 (Cytochromes c)
EC 1.7.- (Nitrite Reductases)
EC 1.7.2.1 (nitrite reductase, copper-containing)

Date Created: 20200408 Date Completed: 20210617 Latest Revision: 20210617

20250114

10.1111/febs.15324

32255260