Human prostaglandin reductase 1 (PGR1): Substrate specificity, inhibitor analysis and site-directed mutagenesis

Chem Biol Interact. 2015 Jun 5;234:105-13. doi: 10.1016/j.cbi.2015.01.021. Epub 2015 Jan 22.

Abstract

Prostaglandins (PGs) are lipid compounds derived from arachidonic acid by the action of cyclooxygenases, acting locally as messenger molecules in a wide variety of physiological processes, such as inflammation, cell survival, apoptosis, smooth muscle contraction, adipocyte differentiation, vasodilation and platelet aggregation inhibition. In the inactivating pathway of PGs, the first metabolic intermediates are 15-keto-PGs, which are further converted into 13,14-dihydro-15-keto-PGs by different enzymes having 15-keto-PG reductase activity. Three human PG reductases (PGR), zinc-independent members of the medium-chain dehydrogenase/reductase (MDR) superfamily, perform the first irreversible step of the degradation pathway. We have focused on the characterization of the recombinant human enzyme prostaglandin reductase 1 (PGR1), also known as leukotriene B4 dehydrogenase. Only a partial characterization of this enzyme, isolated from human placenta, had been previously reported. In the present work, we have developed a new HPLC-based method for the determination of the 15-keto-PG reductase activity. We have performed an extensive kinetic characterization of PGR1, which catalyzes the NADPH-dependent reduction of the α,β-double bond of aliphatic and aromatic aldehydes and ketones, and 15-keto-PGs. PGR1 also shows low activity in the oxidation of leukotriene B4. The best substrates in terms of kcat/Km were 15-keto-PGE2, trans-3-nonen-2-one and trans-2-decenal. Molecular docking simulations, based on the three-dimensional structure of the human enzyme (PDB ID 2Y05), and site-directed mutagenesis studies were performed to pinpoint important structural determinants, highlighting the role of Arg56 and Tyr245 in 15-keto-PG binding. Finally, inhibition analysis was done using non-steroidal anti-inflammatory drugs (NSAIDs) as potential inhibitors.

Keywords: Alkenal/one-oxidoreductase; Leukotriene B4 12-hydroxy-dehydrogenase; Medium-chain dehydrogenase/reductase; Non-steroidal anti-inflammatory drug; Prostaglandin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehydes / metabolism
  • Alkenes / metabolism
  • Amino Acid Sequence
  • Dinoprostone / analogs & derivatives
  • Dinoprostone / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics*
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Ketones / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed / methods
  • NADP / metabolism
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism*
  • Oxidoreductases / metabolism
  • Sequence Alignment
  • Substrate Specificity / genetics*

Substances

  • (2E)-decenal
  • Aldehydes
  • Alkenes
  • Intracellular Signaling Peptides and Proteins
  • Ketones
  • MRFAP1 protein, human
  • Nuclear Proteins
  • 15-ketoprostaglandin E2
  • NADP
  • Oxidoreductases
  • Dinoprostone