ABSTRACT
Two acidic residues, Glu-48 and Glu-49, of cytochrome b5 (b5) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Substitution of Ala, Gly, Cys, or Gln for these two glutamic acid residues abrogated all capacity to stimulate 17,20-lyase activity. Mutations E49D and E48D/E49D retained 23 and 38% of wild-type activity, respectively. Using the zero-length cross-linker ethyl-3-(3-dimethylaminopropyl)carbodiimide, we obtained cross-linked heterodimers of b5 and CYP17A1, wild-type, or mutations R347K and R358K. In sharp contrast, the b5 double mutation E48G/E49G did not form cross-linked complexes with wild-type CYP17A1. Mass spectrometric analysis of the CYP17A1-b5 complexes identified two cross-linked peptide pairs as follows: CYP17A1-WT: (84)EVLIKK(89)-b5: (53)EQAGGDATENFEDVGHSTDAR(73) and CYP17A1-R347K: (341)TPTISDKNR(349)-b5: (40)FLEEHPGGEEVLR(52). Using these two sites of interaction and Glu-48/Glu-49 in b5 as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the CYP17A1-b5 complex. The appositional surfaces include Lys-88, Arg-347, and Arg-358/Arg-449 of CYP17A1, which interact with Glu-61, Glu-42, and Glu-48/Glu-49 of b5, respectively. Our data reveal the structural basis of the electrostatic interactions between these two proteins, which is critical for 17,20-lyase activity and androgen biosynthesis.
Subject(s)
Amino Acids/chemistry , Cytochromes b5/chemistry , Steroid 17-alpha-Hydroxylase/chemistry , Amino Acid Sequence , Amino Acids/metabolism , Catalytic Domain , Cross-Linking Reagents/chemistry , Crystallography, X-Ray , Cytochromes b5/classification , Cytochromes b5/genetics , Cytochromes b5/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Ethyldimethylaminopropyl Carbodiimide/chemistry , Gene Expression , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/classification , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , Steroid 17-alpha-Hydroxylase/classification , Steroid 17-alpha-Hydroxylase/genetics , Steroid 17-alpha-Hydroxylase/metabolism , ThermodynamicsABSTRACT
Desaturases that introduce double bonds into the fatty acids are involved in the adaptation of membrane fluidity to changes in the environment. Besides, polyunsaturated fatty acids (PUFAs) are increasingly recognized as important pharmaceutical and nutraceutical compounds. To successfully engineer organisms with increased stress tolerance or the ability to synthesize valuable PUFAs, detailed knowledge about the complexity of the desaturase family as well as understanding of the coevolution of desaturases and their cytochrome b5 electron donors is needed. We have constructed phylogenies of several hundred desaturase sequences from animals, plants, fungi and bacteria and of the cytochrome b5 domains that are fused to some of these enzymes. The analysis demonstrates the existence of three major desaturase acyl-CoA groups that share few similarities. Our results indicate that the fusion of Delta(6)-desaturase-like enzymes with their cytochrome b5 electron donor was a single event that took place in the common ancestor of all eukaryotes. We also propose the Delta(6)-desaturase-like enzymes as the most probable donor of the cytochrome b5 domain found in fungal Delta(9)-desaturases and argue that the recombination most likely happened soon after the separation of the animal and fungal ancestors. These findings answer some of the previously unresolved questions and contribute to the quickly expanding field of research on desaturases.
Subject(s)
Cytochromes b5/classification , Eukaryota/enzymology , Fatty Acid Desaturases/classification , Phylogeny , Amino Acid Sequence , Animals , Computational Biology , Cytochromes b5/chemistry , Cytochromes b5/genetics , Fatty Acid Desaturases/chemistry , Fatty Acid Desaturases/genetics , Fatty Acids, Unsaturated/metabolism , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Bax inhibitor-1 (BI-1) is a widely conserved cytoprotective protein localized in the endoplasmic reticulum (ER) membrane. We identified Arabidopsis cytochrome b(5) (AtCb5) as an interactor of Arabidopsis BI-1 (AtBI-1) by screening the Arabidopsis cDNA library with the split-ubiquitin yeast two-hybrid (suY2H) system. Cb5 is an electron transfer protein localized mainly in the ER membrane. In addition, a bimolecular fluorescence complementation (BiFC) assay and fluorescence resonance energy transfer (FRET) analysis confirmed that AtBI-1 interacted with AtCb5 in plants. On the other hand, we found that the AtBI-1-mediated suppression of cell death in yeast requires Saccharomyces cerevisiae fatty acid hydroxylase 1 (ScFAH1), which had a Cb5-like domain at the N terminus and interacted with AtBI-1. ScFAH1 is a sphingolipid fatty acid 2-hydroxylase localized in the ER membrane. In contrast, AtFAH1 and AtFAH2, which are functional ScFAH1 homologues in Arabidopsis, had no Cb5-like domain, and instead interacted with AtCb5 in plants. These results suggest that AtBI-1 interacts with AtFAHs via AtCb5 in plant cells. Furthermore, the overexpression of AtBI-1 increased the level of 2-hydroxy fatty acids in Arabidopsis, indicating that AtBI-1 is involved in fatty acid 2-hydroxylation.
