PDBsum entry 3h4h

Oxidoreductase

PDB id: 3h4h

Contents

Protein chains

335 a.a. *

Metals

_CU ×6

Waters ×1344

* Residue conservation analysis

PDB id:

3h4h

Name:

Oxidoreductase

Title:

Met94thr/phe312cys variant of nitrite reductase from alcaligenes faecalis

Structure:

Copper-containing nitrite reductase. Chain: a, b, c. Synonym: cu-nir. Engineered: yes

Source:

Alcaligenes faecalis. Organism_taxid: 511. Strain: s-6. Gene: nir, nirk. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

1.60Å R-factor: 0.183 R-free: 0.226

Authors:

I.S.Macpherson,I.S.Murphy

Key ref:

I.S.MacPherson et al. (2010). Directed evolution of copper nitrite reductase to a chromogenic reductant. Protein Eng Des Sel, 23, 137-145. PubMed id: 20083495

Date:

20-Apr-09 Release date: 02-Feb-10

Protein chains

P38501  (NIR_ALCFA) -  Copper-containing nitrite reductase from Alcaligenes faecalis

Seq: 376 a.a.

Struc: 335 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.7.2.1 - nitrite reductase (NO-forming).
• Reaction: nitric oxide + Fe(III)-[cytochrome c] + H2O = Fe(II)-[cytochrome c] + nitrite + 2 H⁺
• Cofactor: Cu cation or Fe cation; FAD

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

Protein Eng Des Sel 23:137-145 (2010)

PubMed id: 20083495

Directed evolution of copper nitrite reductase to a chromogenic reductant.

I.S.MacPherson, F.I.Rosell, M.Scofield, A.G.Mauk, M.E.Murphy.

ABSTRACT

Directed evolution methods were developed for Cu-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6. The PCR cloning strategy allows for the efficient production of libraries of 100 000 clones by a modification of a megaprimer-based whole-plasmid synthesis reaction. The high-throughput screen includes colony lift onto a nylon membrane and subsequent lysis of NiR-expressing colonies in the presence of Cu(2+) ions for copper incorporation into intracellularly expressed NiR. Addition of a chromogenic substrate, 3, 3'-diaminobenzidine (DAB), results in deposition of red, insoluble color at the site of oxidation by functional NiR. Twenty-thousand random variants of NiR were screened for improved function with DAB as a reductant, and five variants were identified. These variants were shuffled and screened, yielding two double variants. An analog of the DAB substrate, o-dianisidine, which is oxidized to a water-soluble product was used for functional characterization. The double variant M150L/F312C was most proficient at o-dianisidine oxidation with dioxygen as the electron acceptor (5.5X wt), and the M150L single variant was most proficient at o-dianisidine oxidation with nitrite as the electron acceptor (8.5X wt). The library generation and screening method can be employed for evolving new reductase functions in NiR and for screening of efficient folding of engineered NiRs.