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

Daryl D. Meling, Susan Zelasko, Amogh Kambalyal, Jahnabi Roy, Aditi Das

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)34-40
Number of pages7
JournalBiophysical Chemistry
Volume200-201
DOIs
StatePublished - Jul 1 2015

Keywords

  • CYP5A1
  • Cytochrome P450
  • Eicosanoids
  • Nanodiscs
  • Protein engineering
  • Thromboxane synthase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Organic Chemistry

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