title: Functional role of the conserved i-helix residue I346 in CYP5A1-Nanodiscs.
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.
authors.affiliation: Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana IL 61802, USA.
authors.forename: Daryl D
authors.initials: DD
authors.lastname: Meling
authors.affiliation: Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana IL 61802, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Zelasko
authors.affiliation: Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana IL 61802, USA.
authors.forename: Amogh
authors.initials: A
authors.lastname: Kambalyal
authors.affiliation: Department of Chemistry, University of Illinois Urbana-Champaign, IL 61802, USA.
authors.forename: Jahnabi
authors.initials: J
authors.lastname: Roy
authors.affiliation: Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana IL 61802, USA; Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana IL 61802, USA; Beckman Institute for Advanced Science and Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana IL 61802, USA. Electronic address: aditidas@illinois.edu.
authors.forename: Aditi
authors.initials: A
authors.lastname: Das
mesh_terms: D017124:Conserved Sequence / Q000235:genetics; D006801:Humans; D007532:Isoleucine / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D049329:Nanostructures / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering; D013930:Thromboxane-A Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D007532:Isoleucine; D013930:Thromboxane-A Synthase
keywords: CYP5A1; Cytochrome P450; Eicosanoids; Nanodiscs; Protein engineering; Thromboxane synthase