ABSTRACT
Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack chalcone isomerase activity and also the fatty acid-binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS AND NOMENCLATURE
- CHI
- chalcone isomerase
- CHIL
- chalone isomerase like
- FAP
- fatty acid binding protein
- DNA
- deoxyribonucleic acid
- HMG-CoA
- β-Hydroxy β-methylglutaryl-CoA
- Ndxx
- N-terminal deletion of xx amino acids
- ORF
- open reading frame
- SC
- synthetic complete
- Ura-
- uracil drop-out media
- GPD
- glyceraldehyde-3-phosphate dehydrogenase
- SS
- salmon sperm DNA
- PEG
- polyethylene glycol
- FLAG
- immunoprecipitation epitope consisting of Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
- bp
- base pairs
- SDM
- site-directed mutagenesis
- BME
- β-mercaptoethanol
- TCA
- trichloroacetic acid
- LDS
- lithium dodecyl sulfate
- SDS-PAGE
- sodium dodecyl sulfate–polyacrylamide gel electrophoresis
- PVDF
- polyvinylidene fluoride
- NFDM
- non-fat dry milk
- TBST
- tris buffered saline plus 0.05% tween-20 (v/v)
- (v/v)
- percent volume per 100 mL volume
- (w/v)
- percent weight per 100 mL volume
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- CYTB
- cytochrome b
- CIT
- citrate synthase
- TOM
- translocase of the outer membrane
- TIM
- translocase of the inner membrane
- SEC
- size-exclusion chromatography
- TCEP
- tris(2-carboxyethyl)phosphine
- DTT
- dithiothreitol
- DSF
- differential scanning fluorimetry
- UV-Vis
- ultraviolet–visible spectroscopy
- CoQ
- coenzyme Q or ubiquinone
- DMQ
- demethoxyubiquinone
- PPHB
- polyprenyl-4-hydroxybenzoate
- PDB
- Protein Data Bank
