title: Characterization of an Additional Splice Acceptor Site Introduced into CYP4B1 in Hominoidae during Evolution.
abstract: CYP4B1 belongs to the cytochrome P450 family 4, one of the oldest P450 families whose members have been highly conserved throughout evolution. The CYP4 monooxygenases typically oxidize fatty acids to both inactive and active lipid mediators, although the endogenous ligand(s) is largely unknown. During evolution, at the transition of great apes to humanoids, the CYP4B1 protein acquired a serine instead of a proline at the canonical position 427 in the meander region. Although this alteration impairs P450 function related to the processing of naturally occurring lung toxins, a study in transgenic mice suggested that an additional serine insertion at position 207 in human CYP4B1 can rescue the enzyme stability and activity. Here, we report that the genomic insertion of a CAG triplet at the intron 5-exon 6 boundary in human CYP4B1 introduced an additional splice acceptor site in frame. During evolution, this change occurred presumably at the stage of Hominoidae and leads to two major isoforms of the CYP4B1 enzymes of humans and great apes, either with or without a serine 207 insertion (insSer207). We further demonstrated that the CYP4B1 enzyme with insSer207 is the dominant isoform (76%) in humans. Importantly, this amino acid insertion did not affect the 4-ipomeanol metabolizing activities or stabilities of the native rabbit or human CYP4B1 enzymes, when introduced as transgenes in human primary cells and cell lines. In our 3D modeling, this functional neutrality of insSer207 is compatible with its predicted location on the exterior surface of CYP4B1 in a flexible side chain. Therefore, the Ser207 insertion does not rescue the P450 functional activity of human CYP4B1 that has been lost during evolution.
authors.affiliation: Department of Pediatric Hematology, Oncology and Clinical Immunology, Children's Hospital, Heinrich Heine University, 40225 Düsseldorf, Germany.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Schmidt
authors.affiliation: Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich Heine University, 40225 Düsseldorf, Germany.
authors.forename: Constanze
authors.initials: C
authors.lastname: Wiek
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA 98195, United States of America.
authors.forename: Oliver T
authors.initials: OT
authors.lastname: Parkinson
authors.affiliation: Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich Heine University, 40225 Düsseldorf, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Roellecke
authors.affiliation: Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich Heine University, 40225 Düsseldorf, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Freund
authors.affiliation: Department of Pediatric Hematology, Oncology and Clinical Immunology, Children's Hospital, Heinrich Heine University, 40225 Düsseldorf, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Gombert
authors.affiliation: Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich Heine University, 40225 Düsseldorf, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Lottmann
authors.affiliation: Wellcome Trust Sanger Institute, Cambridge, United Kingdom.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Steward
authors.affiliation: Division of Pediatric Hematology and Oncology, Department of Child and Adolescent Health, University of Göttingen, 37099 Göttingen, Germany.
authors.forename: Christof M
authors.initials: CM
authors.lastname: Kramm
authors.affiliation: Departments of Physiology and Membrane Biology, University of California Davis, Davis, CA 95616, United States of America.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Yarov-Yarovoy
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA 98195, United States of America.
authors.forename: Allan E
authors.initials: AE
authors.lastname: Rettie
authors.affiliation: Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich Heine University, 40225 Düsseldorf, Germany; Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, United States of America; Department of Pediatrics III, University Children's Hospital Essen, University of Duisburg-Essen, 45122 Essen, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: Hanenberg
mesh_terms: D000328:Adult; D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics*; D005075:Biological Evolution; D016923:Cell Death / Q000187:drug effects; D004795:Enzyme Stability; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D008958:Models, Molecular; D016254:Mutagenesis, Insertional / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics*; D011817:Rabbits; D012694:Serine / Q000235:genetics; D013729:Terpenes / Q000494:pharmacology
chemical_list: D022821:RNA Splice Sites; D013729:Terpenes; D012694:Serine; D001189:Aryl Hydrocarbon Hydroxylases; C095029:cytochrome P-450 CYP4B1; C001470:4-ipomeanol