ABSTRACT
The comparative transcriptome analysis of the fungus Gibberella zeae which could efficiently catalyze the 7ß-hydroxylation of LCA to produce UDCA was performed with LCA induction. This is the first time to report the comparative transcriptome of fungus under LCA treatment. Totally, 1364 differentially expressed genes including 770 up-regulated and 594 down-regulated genes were identified. In the 770 up-regulated genes, 12 genes with the function of hydroxylation were picked out by application of function screening, which were annotated as CYP450 or hydroxylase. Moreover, the qRT-PCR results of five up-regulated CYP450-like genes confirmed the credibility of RNA-Seq further. These results provide valuable information for the discovery of novel enzyme producing clinical drug UDCA from butchery byproduct LCA, and also might indicate some clues for the detoxification process of LCA in humans.
Subject(s)
Fusarium/genetics , Lithocholic Acid/metabolism , Transcriptome/genetics , Ursodeoxycholic Acid/metabolism , Gene Expression Regulation, Fungal/genetics , Humans , Lithocholic Acid/genetics , Mixed Function Oxygenases/genetics , Retinoic Acid 4-Hydroxylase/genetics , Ursodeoxycholic Acid/geneticsABSTRACT
Lithocholate (LC) (10-300 microM) in physiological solution is sensed by vascular myocyte large conductance, calcium- and voltage-gated potassium (BK) channel beta(1) accessory subunits, leading to channel activation and arterial dilation. However, the structural features in steroid and target that determine LC action are unknown. We tested LC and close analogs on BK channel (pore-forming cbv1+beta(1) subunits) activity using the product of the number of functional ion channels in the membrane patch (N) and the open channel probability (Po). LC (5beta-cholanic acid-3alpha-ol), 5alpha-cholanic acid-3alpha-ol, and 5beta-cholanic acid-3beta-ol increased NPo (EC(50) approximately 45 microM). At maximal increase in NPo, LC increased NPo by 180%, whereas 5alpha-cholanic acid-3alpha-ol and 5beta-cholanic acid-3beta-ol raised NPo by 40%. Thus, the alpha-hydroxyl and the cis A-B ring junction are both required for robust channel potentiation. Lacking both features, 5alpha-cholanic acid-3beta-ol and 5-cholenic acid-3beta-ol were inactive. Three-dimensional structures show that only LC displays a bean shape with clear-cut convex and concave hemispheres; 5alpha-cholanic acid-3alpha-ol and 5beta-cholanic acid-3beta-ol partially matched LC shape, and 5alpha-cholanic acid-3beta-ol and 5-cholenic acid-3beta-ol did not. Increasing polarity in steroid rings (5beta-cholanic acid-3alpha-sulfate) or reducing polarity in lateral chain (5beta-cholanic acid 3alpha-ol methyl ester) rendered poorly active compounds, consistent with steroid insertion between beta(1) and bilayer lipids, with the steroid-charged tail near the aqueous phase. Molecular dynamics identified two regions in beta(1) transmembrane domain 2 that meet unique requirements for bonding with the LC concave hemisphere, where the steroid functional groups are located.
