Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

Abstract

The three‐dimensional structure of recombinant homodimeric delta9 stearoyl‐acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6‐fold non‐crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha‐helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di‐iron centre, with ligands from four of the alpha‐helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu‐oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions.

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase,

“…Given that the geometries of the active site metal clusters are most similar to that of the mixed-valent Fe(II)Fe(III) form of MMOH (49), one possibility is that photoreduction by synchrotron radiation occurred to produce the mixedvalent oxidation state. A similar phenomenon has been reported previously for structures of Δ 9 -desaturase and RNR-R2, although the oxidized forms of these proteins were reduced by two electrons (53)(54)(55). The structures of the PHH diiron centers presented here are not chargebalanced, however, and would require an additional negatively charged ligand, such as a bridging hydroxide ion, to achieve a neutral state similar to that observed in MMOH mv ( Figure 2C).…”

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

“…The Zn(II)-Zn(II) distance is 3.9 Å, close to the distance observed between metal ions in the di-Mn(II) forms of bacterioferritin (4.0 Å) and R2 (3.6 -3.7 Å), the diferrous forms of ⌬ 9 ACP desaturase (4.2 Å) and ribonucleotide reductase R2 (3.8 Å), and the Fe-Zn(II) form of rubrerythrin (3.7 Å) (68,69). The pentacoordinate ligand arrangement with two 1,3 bridging carboxylates and two chelating carboxylates is also nearly identical to that observed in di-Fe(II) ⌬ 9 ACP desaturase (71) and di-Mn(II) bacterioferritin (67). In these proteins, the ligands surround the metal ions except for a vacant pair of sites along one face of the dimetal center.…”

“…A second, functionally diverse class of carboxylate-bridged diiron proteins (2-4, 65) features a helical Glu-Xxx-Xxx-His (EXXH) motif, with two rather than three residues between the liganding side chains. We have conducted a retrostructural analysis (27) of six members of this class, including three ferroxidases [ferritin (66), bacterioferritin (67), and rubrerythrin (68, 69)], ribonucleotide reductase R2 subunit (R2) (70), ⌬ 9 ACP desaturase (71), and the catalytic subunit of methane monooxygenase (72). Although there is less than 5% sequence identity common to all members of this class, their active sites are housed within a very simple pseudo-222-symmetric four-helix bundle.…”

Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate,

“…The binding of 18:0-ACP to Δ9D is a critical step in the catalytic cycle, because it serves to activate the enzyme's diiron cluster for O 2 binding (10) and, in chemically-reduced Δ9D, prompts formation of a stable peroxodiferric state for the iron cluster (11). A deep hydrophobic groove in Δ9D revealed by X-ray crystallography (12) has been suggested as the fatty acid binding site. This hypothesis is supported by studies showing that ACPs with shorter acyl chains bind more weakly to Δ9D (13).…”