PDBsum entry 2flq

Oxidoreductase

PDB id: 2flq

Contents

Protein chains

359 a.a. *

Ligands

ARG ×2

HEM ×2

Waters ×223

* Residue conservation analysis

PDB id:

2flq

Name:

Oxidoreductase

Title:

Crystal structure of nitric oxide synthase from geobacillus stearothermophilus (atcc 12980) complexed with l-arginine

Structure:

Nitric oxide synthase. Chain: a, b. Engineered: yes

Source:

Geobacillus stearothermophilus. Organism_taxid: 1422. Strain: atcc 12980. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PQS)

Resolution:

3.20Å R-factor: 0.222 R-free: 0.295

Authors:

J.Sudhamsu,B.R.Crane

Key ref:

J.Sudhamsu and B.R.Crane (2006). Structure and reactivity of a thermostable prokaryotic nitric-oxide synthase that forms a long-lived oxy-heme complex. J Biol Chem, 281, 9623-9632. PubMed id: 16407211 DOI: 10.1074/jbc.M510062200

Date:

06-Jan-06 Release date: 31-Jan-06

Protein chains

Q5KZC5  (Q5KZC5_GEOKA) -  Nitric oxide synthase from Geobacillus kaustophilus (strain HTA426)

Seq: 440 a.a.

Struc: 359 a.a.*

* PDB and UniProt seqs differ at 14 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.14.13.39 - nitric-oxide synthase (NADPH).
• Reaction: 2 L-arginine + 3 NADPH + 4 O2 + H⁺ = 2 L-citrulline + 2 nitric oxide + 3 NADP⁺ + 4 H2O

2 × L-arginine + 3 × NADPH (Bound ligand (Het Group name = ARG) corresponds exactly) + 4 × O2 + H(+) = 2 × L-citrulline + 2 × nitric oxide + 3 × NADP(+) + 4 × H2O

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

reference

DOI no: 10.1074/jbc.M510062200

J Biol Chem 281:9623-9632 (2006)

PubMed id: 16407211

Structure and reactivity of a thermostable prokaryotic nitric-oxide synthase that forms a long-lived oxy-heme complex.

J.Sudhamsu, B.R.Crane.

ABSTRACT

In an effort to generate more stable reaction intermediates involved in substrate oxidation by nitric-oxide synthases (NOSs), we have cloned, expressed, and characterized a thermostable NOS homolog from the thermophilic bacterium Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide (NO) from l-arginine via the stable intermediate N-hydroxy l-arginine (NOHA). The addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of substrates l-arginine and NOHA. The substrate-induced red shift correlates with hydrogen bonding between substrate and heme-bound oxygen resulting in conversion to a ferric heme-superoxy species. In single turnover experiments with NOHA, NO forms only in the presence of H(4)B. The crystal structure of gsNOS at 3.2 AA of resolution reveals great similarity to other known bacterial NOS structures, with the exception of differences in the distal heme pocket, close to the oxygen binding site. In particular, a Lys-356 (Bacillus subtilis NOS) to Arg-365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme. Thus, a more constrained heme pocket may slow ligand dissociation and increase the lifetime of heme-bound oxygen to seconds at 4 degrees C. Similarly, the ferric-heme NO complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise for studying downstream intermediates involved in substrate oxidation.

Selected figure(s)

Figure 1.
FIGURE 1. Current mechanistic model for NO biosynthesis by NOS adapted from Stuehr et al. (18). Formation of citrulline, NO, and ferric heme marks the end of one catalytic cycle. In some mammalian NOS isozymes, further reduction of the ferric-heme nitrosyl complex competes with NO release from the active center (18).

Figure 9.
FIGURE 9. Comparison of the active sites of bsNOS (orange) and gsNOS (yellow). In bsNOS, Lys-356 does not interact with Asp-216. A Lys to Arg substitution in gsNOS allows Arg-365 to hydrogen bond with Asp-225 (3.2 Å), altering its side chain position. This change in structure appears to be correlated with movement of Ser-224 that in turn pushes Ile-223 into the active site, reducing the distance between the -carbon of Ile-223 and the heme iron atom from 6.7 Å (bsNOS) to 6.1 Å (gsNOS).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 9623-9632) copyright 2006.

Figures were selected by an automated process.