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Title: Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E. coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the twomore » regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. - Highlights: • Ala62 is located in α-helix A of the carcinogen-metabolizing enzyme CYP1A1. • Pro acts as an α-helix breaker. • A variant protein of CYP1A1, Ala62Pro, had lower heme content than the wild-type. • The variant of CYP1A1 had lower enzyme activities than the wild-type.« less
Authors:
;  [1] ;  [2] ;  [3] ; ;  [4] ;  [1]
  1. College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul (Korea, Republic of)
  2. School of Life Sciences and Biotechnology, Korea University, Seoul (Korea, Republic of)
  3. College of Medicine, Chung-Ang University, Seoul (Korea, Republic of)
  4. College of Agriculture of Life Science, Seoul National University, Seoul (Korea, Republic of)
Publication Date:
OSTI Identifier:
22465769
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 285; Journal Issue: 3; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CARCINOGENS; DEALKYLATION; DETOXIFICATION; DISTURBANCES; EDTA; ENZYME ACTIVITY; ENZYMES; FLUORIDES; HAMSTERS; HEME; HYDROXYLATION; MICROSOMES; MUTAGENESIS; OVARIES; POLLUTANTS; PRECURSOR; PROTEIN STRUCTURE; PYRENE; RESIDUES