PDBsum entry: 1lj1

Oxidoreductase

PDB id: 1lj1

Contents

Protein chains

568 a.a. *

Ligands

HEM ×8

FAD ×2

FUM ×2

Metals

_NA ×2

Waters ×1630

* Residue conservation analysis

PDB id:

1lj1

Name:

Oxidoreductase

Title:

Crystal structure of q363f/r402a mutant flavocytochrome c3

Structure:

Flavocytochrome c3. Chain: a, b. Synonym: fumarate reductase flavoprotein subunit, fcc3. Engineered: yes. Mutation: yes

Source:

Shewanella frigidimarina. Organism_taxid: 56812. Gene: fcc. Expressed in: shewanella frigidimarina. Expression_system_taxid: 56812.

Resolution:

2.00Å R-factor: 0.161 R-free: 0.235

Authors:

C.G.Mowat,K.L.Pankhurst,C.S.Miles,D.Leys,M.D.Walkinshaw, G.A.Reid,S.K.Chapman

Key ref:

C.G.Mowat et al. (2002). Engineering water to act as an active site acid catalyst in a soluble fumarate reductase. Biochemistry, 41, 11990-11996. PubMed id: 12356299 DOI: 10.1021/bi0203177

Date:

18-Apr-02 Release date: 06-Nov-02

Protein chains

P0C278  (FRDA_SHEFR) -  Fumarate reductase flavoprotein subunit

Seq: 571 a.a.

Struc: 568 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.3.99.1 - Succinate dehydrogenase.
• Reaction: Succinate + acceptor = fumarate + reduced acceptor

Succinate (Bound ligand (Het Group name = FUM) corresponds exactly) + acceptor = fumarate + reduced acceptor

• Cofactor: FAD

FAD (Bound ligand (Het Group name = FAD) corresponds exactly)

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

 Gene Ontology (GO) functional annotation 

• Cellular component: intracellular (3 terms)
• Biological process: oxidation reduction (5 terms)
• Biochemical function: electron carrier activity (5 terms)

reference

DOI no: 10.1021/bi0203177

Biochemistry 41:11990-11996 (2002)

PubMed id: 12356299

Engineering water to act as an active site acid catalyst in a soluble fumarate reductase.

C.G.Mowat, K.L.Pankhurst, C.S.Miles, D.Leys, M.D.Walkinshaw, G.A.Reid, S.K.Chapman.

ABSTRACT

The ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, in which it acts both as a Lewis acid in transition-state stabilization and as a Brønsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Brønsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Brønsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site.