The structure of human 5-lipoxygenase - PubMed (original) (raw)

The structure of human 5-lipoxygenase

Nathaniel C Gilbert et al. Science. 2011.

Abstract

The synthesis of both proinflammatory leukotrienes and anti-inflammatory lipoxins requires the enzyme 5-lipoxygenase (5-LOX). 5-LOX activity is short-lived, apparently in part because of an intrinsic instability of the enzyme. We identified a 5-LOX-specific destabilizing sequence that is involved in orienting the carboxyl terminus, which binds the catalytic iron. Here, we report the crystal structure at 2.4 angstrom resolution of human 5-LOX stabilized by replacement of this sequence.

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Figures

Fig 1. Stabilization of human 5-LOX

(A) Superposition of the C-terminal regions of the structures of 15-, 8_R_-, and Stable-5-lipoxygenase. The C-terminal segment that leads to the catalytic Fe emanates from the helix which terminates at amino acid 655 (5-LOX numbering; Stable-5-LOX, pink; 8_R_-LOX green; 15-LOX, blue). Highly conserved amino acids (Leu, Phe/Tyr) and an invariant salt link (Asp-Arg) are depicted in stick rendering. (B) Detail of the turn at the end of the terminal helix. The 5-LOX specific Lys (substituted in Stable-5-LOX with Leu) is modeled at position 655 as its most common rotamer (transparent sphere rendering). As positioned, it would interfere with the invariant salt-link and cation- π interactions. All figures were generated with Pymol (29). (C) Thermal denaturation of Stable-5-LOX (red) and the parent enzyme Sol-5-LOX (blue). Fluorescence (F) is monitored as a function of temperature. Tm (with s.d.) 56.6 (±0.4) and 59.7(±0.2) °C for Sol-5-LOX and Stable-5-LOX, respectively. (D) HPLC chromatogram. Product analysis of Stable-5-LOX reveals both 5-HETE (5-HPETE reduced by the addition of triphenylphosphine, TPP) and Leukotriene A4 hydrolysis products (5,12 diols).

Fig 2. The structure of Stable-5-LOX

(A) A cartoon rendering of 5-LOX. The two views differ by a 180° rotation about the vertical line. The amino terminal C2-like domain is in dark gray, and the catalytic domain in light gray. The distinctive arched helix is in blue, and helix α2 in red. The internal cavity, generated with CastP (30), is in pink and the Fe is an orange sphere. The positions of the mutated amino acids are indicated in mesh rendering: green, putative membrane insertion residues; yellow, proximal cysteines; and blue, the KKK→ENL substitution. (B). Detail of the relationship of the arched helix and helix α2 to the active site as viewed from the perspective indicated by the red arrow in panel (A). Shown in stick rendering are amino acids 406, 414, 420, 421 of the arched helix and F177 and Y181 from helix α2 (with transparent surface rendering). The latter two bulky amino acids obstruct access to the cavity. The proximity of the C-terminal Ile (673) to the corked portal is apparent.

Fig. 3. (A) The positioning of helix α2 is unique in 5-LOX

(A) A 5-LOX cartoon is rendered in pink, 15-LOX in blue and 8_R_-LOX in green. Conserved aromatic amino acids (F169,W201) that flank the region are in stick rendering. F177 and Y181 that make up the “cork” that helps define the active site are in stick. The catalytic iron is an orange sphere. (B) A full overlay of the three structures in which it is apparent that, with the exception of α2, the secondary structural elements in the enzymes are conserved. The box indicates the region amplified in (A).

Fig 4. The 5-LOX active site

Internal cavities calculated with CastP (30). (A) The active site cavity of 15-LOX (2P0M) is in yellow. Invariant Leu and Ile side chains are in green stick rendering. The 5-LOX “FY cork” is superposed on the 15-LOX cavity and plugs the entrance. (B) The equivalent orientation of the active site cavity of Stable-5-LOX in pink; invariant Leu and Ile side chains in green sticks. Note the similarity of the positions of these amino acids to their counterparts in 15-LOX (A). Iron coordination sphere amino acids (C, white) are in stick rendering, and the iron an orange sphere. (C) 5-LOX amino acids that contribute to the active site cavity. Entry into this cavity requires a conformational change.

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The structure of human 5-lipoxygenase - PubMed (original) (raw)