title: Characterization of the biochemical and structural phenotypes of four CYP1B1 mutations observed in individuals with primary congenital glaucoma.
abstract: OBJECTIVE
  The objective of this study was to examine the biochemical and physical properties of cytochrome P450 1B1 (CYP1B1) mutants, test our hypothesis that primary congenital glaucoma (PCG)-causing mutants have altered metabolic activity, and correlate these to structural changes in the molecule.
  
  
  METHODS
  CYP1B1.1 cDNA was mutated to four forms found in individuals with the PCG phenotype, Y81N, E229K, A330F, and R368H. Expression and stability of the mutant hemoproteins and their ability to metabolize beta-estradiol, arachidonic acid, and retinoids, were determined. Alterations in mutant properties were related to structural changes by in silico examination, on the basis of the CYP1A2 crystal structure.
  
  
  RESULTS
  CYP1B1 mutations strongly affected the stability, ease of heterologous expression, and enzymatic properties of the protein. These were related to the location of the amino acid substitutions in the CYP1B1 structure. Three of the mutations involve residues located on the surface of CYP1B1, Y81N, and E229K near the distal surface, and R368H near the proximal surface. The former two substitutions, Y81N and E229K, caused greatly reduced stability at 4 degrees C. Y81N severely inhibited all substrate turnover, but E229K only inhibited arachidonate turnover and exhibited minimal effect on efficiency of retinoid metabolism and estradiol metabolism. The R368H mutation is relatively conservative, affecting charge-pairing with the deeper-located D374, but it severely inhibited metabolism of all substrates tested, and, like Y81N, expression of the enzyme is less facile than CYP1B1wt. The A330F mutation replaces a small alanine by a bulky phenylalanine in the enzyme active site and had major impact on substrate binding, turnover, uncoupling, and metabolite pattern.
  
  
  CONCLUSIONS
  Consistent with the hypothesis, these PCG-related mutations cause identifiable structural changes negatively impacting CYP1B1 biochemistry and stability.
authors.affiliation: Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.
authors.forename: Dharamainder
authors.initials: D
authors.lastname: Choudhary
authors.forename: Ingela
authors.initials: I
authors.lastname: Jansson
authors.forename: Mansoor
authors.initials: M
authors.lastname: Sarfarazi
authors.forename: John B
authors.initials: JB
authors.lastname: Schenkman
mesh_terms: D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli; D004958:Estradiol / Q000378:metabolism; D005901:Glaucoma / Q000151:congenital* / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D017433:Protein Structure, Secondary; D012176:Retinoids / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D012176:Retinoids; D016718:Arachidonic Acid; D004958:Estradiol; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1