Functional role of the conserved i-helix residue I346 in CYP5A1–Nanodiscs

Highlights

• CYP5A1 has an Ile instead of the conserved, I-helix Thr found in most CYPs.

• Mutation of this Ile to a hydroxyl-containing residues Thr or Ser increases TXA₂ formation.

• Spectral changes are noted between the WT and hydroxyl-containing mutants.

Abstract

Thromboxane synthase (CYP5A1) is a non-classical cytochrome P450 (CYP) expressed in human platelets that mediates vascular homeostasis by producing thromboxane A₂ (TXA₂) through the isomerization of prostaglandin H₂ (PGH₂). A homology alignment of CYP5A1 with human CYPs indicates that a highly conserved I-helix threonine residue is occupied by an isoleucine at position 346 in CYP5A1. We find that reverse-engineering CYP5A1 to contain either threonine or serine in this position dramatically increases TXA₂ formation. Interestingly, the levels of malondialdehyde (MDA), a homolytic fragmentation product of PGH₂ formed via a pathway independent of TXA₂ formation, remain constant. Furthermore, spectral analysis using two PGH₂ substrate analogs supports the observed activity changes in the hydroxyl-containing mutants. The more constrained active site of the I346T mutant displays altered PGH₂ substrate analog binding properties. Together these studies provide new mechanistic insights into CYP5A1 mediated isomerization of PGH₂ with respect to a critical active site residue.

Introduction

Cytochrome P450s (CYPs) are heme-containing enzymes that catalyze numerous oxidative reactions in nature including the hydroxylation and epoxidation of alkenes, as well as less commonly, the isomerization of endoperoxide-containing substrates [1]. In order to catalyze hydroxylation or epoxidation reactions, classical Type I and II CYPs shuttle through heme–oxygen reactive intermediates such as iron–hydroperoxo and iron–oxenoid species (Supplementary Fig. S1). This hydroperoxo intermediate of the heme is thought to be stabilized by the hydroxyl group of a highly conserved threonine residue in the I-helix, positioned on the distal side of the heme active site (Supplementary Fig. S1 and S2) [2], [3]. This residue mediates the formation of an appropriate hydrogen bonding network, either directly by the threonine group or by the positioning of a water molecule within the active site [3]. Previous mutagenesis studies on this threonine residue in other CYPs have shown that mutating this residue significantly impacts enzyme function. The alteration of this residue presumably destabilizes the iron–hydroperoxo intermediate, leading to a decrease in formation of the native hydroxylated or epoxygenated product and an increase in the production of reactive oxygen species [3], [4], [5], [6], [7]. The hydrogen bonding network facilitated by this threonine is also important in many CYPs for two proton transfer steps that allow the heme intermediate to transition from an iron–peroxo to an iron–hydroperoxo and then to an iron–oxenoid species, eventually leading to the epoxidation or hydroxylation of the substrate [8].

Interestingly, a sequence homology alignment of the known human CYPs show that there are a few human CYPs that lack this conserved threonine and instead contain isoleucine or asparagine (Supplementary Fig. S2 and S3). These CYPs are either isomerases (e.g. CYP5A1, CYP8A1) instead of typical hydroxylases or they have unknown functions (e.g. CYP20A1, CYP39A). Prostacyclin synthase (CYP8A1) has an asparagine (N287) residue instead of threonine and the CYP8A1 crystal structure suggests that the capacity to form a hydrogen-bond network through the asparagine remains [9]. In contrast, an isoleucine residue is present in two human CYPs, thromboxane synthase (CYP5A1) and the orphan CYP20A1, as well as in allene oxide synthase (CYP74A1) from the plant Arabidopsis thaliana [[10], [11]]. Isoleucine is incapable of participating in a hydrogen bonding network and the purpose of this active site residue in product formation has not been thoroughly examined.

Of the two human CYPs with an isoleucine instead of a threonine, CYP5A1 has been studied more extensively and is known to mediate cardiovascular homeostasis by generating thromboxane A₂ (TXA₂). CYP5A1 is expressed in platelets [12] and converts prostaglandin H₂ (PGH₂) either into TXA₂ via an isomerization of the endoperoxide bond or into two fragmentation products, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA) (Fig. 1) [13], [14].

HHT is an endogenous ligand for the leukotriene B4 receptor BLT2 which is implicated in allergic airway inflammation [16]. The precise role of MDA remains undetermined, however, it has been found to form adducts with proteins, phospholipids [17], and DNA, particularly in atherosclerotic lesions [18]. Homeostatic imbalances in CYP5A1 that lead to the over-production of TXA₂ have been implicated in the development of several major disorders [19], [20], [21], [22]. Owing to the unique biochemical nature and physiological significance of CYP5A1, it is important to better understand the mechanistic details of this enzyme with respect to this specific residue Ile346.

In this work, we have reverse-engineered the native isoleucine 346 to threonine in CYP5A1 in order to understand its role in substrate binding and product formation. We have also mutated this isoleucine 346 to serine, a smaller hydroxyl-containing residue, to examine the spatial constraints of the substrate orientation within the enzyme active site. Here we describe the biochemical characteristics of these mutants and show that the introduction of a hydroxyl-containing residue significantly influences native CYP5A1 function. All studies were performed using CYP5A1 in Nanodiscs (i.e. nanoscale lipid bilayers) that stabilize the enzyme in a native membrane environment. This produces reproducible and cleaner spectroscopic data and provides a native-like membrane environment to evaluate the enzyme function [23], [24], [25], [26], [27], [28], [29], [30]. Hence, this work provides insights into the role of the key active site residue 346 in thromboxane synthase function within a native membrane environment with respect to substrate isomerization and substrate analog binding.