Leukotriene A4 hydrolase : studies of structure-function relationships by site-directed mutagenesis and X-ray crystallography

Inflammation is the pathophysiological mechanism by which an organism clears and rebuilds a site of infection or injury. It is also the characteristic feature of a multitude of severe diseases, with the potential of afflicting almost any organ system. The inflammatory process is a complex array of interactions between cells and soluble factors, among which are the leukotrienes, a family of lipid mediators.

Leukotriene (LT) B4 is a chemical mediator of inflammation. It is a potent chemotactic agent for polymorphonuclear leukocytes and T-lymphocytes, and thus constitutes an important link between the innate and adaptive immune responses. LTB4 is formed from arachidonic acid by the sequential action of 5-lipoxygenase and LTA4 hydrolase (LTA4H), which yield the unstable epoxide intermediate LTA4 and LT134, respectively. LTA4H is a ubiquitous 69 kDa dual-activity zinc metalloenzyme. In addition to the epoxide hydrolase activity, i.e. the conversion of LTA4 into LT134, it exhibits an anion-dependent aminopeptidase activity, for which the physiologic substrate(s) is currently not known. LTA4H belongs to the MI family of metallopeptidases and shares conserved sequence motifs with members of this family.

Inhibition studies and the recently solved crystal structure of LTA4H showed that the epoxide hydrolase and aminopeptidase reactions occur in a common active site. For the aminopeptidase reaction, certain structural determinants had been characterized, but for the epoxide hydrolase reaction, the catalytic determinants in LTA4H were largely unknown. In this thesis we present studies of structure-function relationships in LTA4H using site-directed mutagenesis and X-ray crystallography.

A conserved GXMEN motif was identified in sequence alignments of members of the M1 family of metallopeptidases. In addition, in the LTA4H crystal structure, Glu-271 of the GXMEN motif seemed to interact with the free amine of the inhibitor bestatin. We show that Glu-271 is crucial for both catalytic reactions of LTA4H, and support these findings with the crystal structure of [E271 Q]LTA4H. For the aminopeptidase reaction, Glu-271 acts as an N-terminal recognition site for peptide substrates. For the epoxide hydrolase reaction, we propose a reaction mechanism in which Glu-271 assists the catalytic zinc in the opening of the substrate's epoxide. Hence, Glu-271 is a unique example of an amino acid residue with dual and distinct functions in two catalytic reactions, involving chemically diverse substrates.

The 12(R)-hydroxyl group is required for the biologic activity of LT134- In the LTA4H crystal structure, a group of hydrophilic residues was identified in predominantly lipophilic surroundings in the active site. The polar side-groups of these residues were catalytic candidates for substrate hydroxylation. We show that mutagenetic exchange of Asp-375 selectively eliminates the epoxide hydrolase reaction and support the critical role of Asp-375 with crystallographic data of [D375N]LTA4H. We propose a mechanism model, in which this residue acts as a general base in substrate 12(R)-hydroxylation. In this role, Asp-375 is a critical element for LTB4 formation and bioactivity.

In the LTA4H crystal structure, the carboxylate of the complexed inhibitor bestatin is located in vicinity of Arg-563 and Lys-565. When the substrate LTA4 is modelled in the active site, electrostatic interactions seem possible between its carboxylate and these two residues. Our mutagenetic data suggest that Arg-563 alone plays a critical role for substrate alignment in the epoxide hydrolase reaction, whereas for the aminopeptidase reaction, Arg-563 cooperates with Lys565 for substrate alignment and binding. These findings are supported by the [R563A]LTA4H crystal structure. Together, these residues constitute a common carboxylate recognition site, with distinct roles for the two reaction mechanisms in LTA4H.

Based on the crystal structure and the characterization of active site catalytic residues, we aimed to design novel inhibitors of LTA4H by computer-assisted molecular modelling and automated docking. We present the design, synthesis and analysis of a novel inhibitor designated HACAP, with IC50 values in the nanomolar range towards both the aminopeptidase and epoxide hydrolase reaction of LTA4H. Furthermore, protein-ligand interactions are confirmed by the LTA4H-HACAP crystal structure complex at 2.0 A resolution.

List of scientific papers

I. Rudberg PC, Tholander F, Thunnissen MM, Haeggstrom JZ (2002). "Leukotriene A4 hydrolase/aminopeptidase. Glutamate 271 is a catalytic residue with specific roles in two distinct enzyme mechanisms. " J Biol Chem 277(2): 1398-404. Epub 2001 Oct 23
https://pubmed.ncbi.nlm.nih.gov/11675384

II. Rudberg PC, Tholander F, Thunnissen MM, Samuelsson B, Haeggstrom JZ (2002). "Leukotriene A4 hydrolase: selective abrogation of leukotriene B4 formation by mutation of aspartic acid 375. " Proc Natl Acad Sci U S A 99(7): 4215-20. Epub 2002 Mar 26
https://pubmed.ncbi.nlm.nih.gov/11917124

III. Rudberg PC, Tholander F, Andberg M, Thunnissen MM, Haeggstrom JZ (2004). "Leukotriene A4 hydrolase: identification of a common carboxylate recognition site for the epoxide hydrolase and aminopeptidase substrates." J Biol Chem 279(26): 27376-82. Epub 2004 Apr 12
https://pubmed.ncbi.nlm.nih.gov/15078870

IV. Rudberg PC, Duron S, Muroya A, Thunnissen MM, Wong CH, Haeggstrom JZ (2004). "HACAP - a novel inhibitor of leukotriene A4 hydrolase: Design, synthesis and structure of the enzyme-inhibitor complex." (Manuscript)