phenanthrenes and Fatty-Liver

A gene encoding a functionally unknown protein that is specifically expressed in the thyroidectomized chicken fatty liver and has a predicted amino acid sequence similar to that of NAD(P)H-dependent carbonyl reductase was overexpressed in Escherichia coli; its product was purified and characterized. The expressed enzyme was an NAD(P)H-dependent broad substrate specificity carbonyl reductase and was inhibited by arachidonic acid at 1.5 μm. Enzymological characterization indicated that the enzyme could be classified as a cytosolic-type carbonyl reductase. The enzyme's 3D structure was determined using the molecular replacement method at 1.98 Å resolution in the presence of NADPH and ethylene glycol. The asymmetric unit consisted of two subunits, and a noncrystallographic twofold axis generated the functional dimer. The structures of the subunits, A and B, differed from each other. In subunit A, the active site contained an ethylene glycol molecule absent in subunit B. Consequently, Tyr172 in subunit A rotated by 103.7° in comparison with subunit B, which leads to active site closure in subunit A. In Y172A mutant, the Km value for 9,10-phenanthrenequinone (model substrate) was 12.5 times higher than that for the wild-type enzyme, indicating that Tyr172 plays a key role in substrate binding in this carbonyl reductase. Because the Tyr172-containing active site lid structure (Ile164-Gln174) is not conserved in all known carbonyl reductases, our results provide new insights into substrate binding of carbonyl reductase. The catalytic properties and crystal structure revealed that thyroidectomized chicken fatty liver carbonyl reductase is a novel enzyme.

Topics: Aldehyde Reductase; Aldo-Keto Reductases; Amino Acid Sequence; Amino Acid Substitution; Animals; Biocatalysis; Catalytic Domain; Chickens; Databases, Protein; Disease Models, Animal; Fatty Liver; Gene Expression Regulation, Enzymologic; Hypothyroidism; Liver; Molecular Sequence Data; Mutant Proteins; NADP; Phenanthrenes; Protein Conformation; Protein Stability; Protein Subunits; Recombinant Proteins; Tyrosine

Triptolide, a diterpenoid epoxide, is one of the major active ingredients of Tripterygium wilfordii Hook F, a woody vine plant called lei gong teng in China, which is used in traditional Chinese Medicine (TCM) for treating many diseases. In this paper, we investigate the relation between inhibition of mitochondrial respiratory chain and liver injury induced by triptolide. Results indicate that the secondary β-oxidation impairment caused by inhibition of mitochondrial respiratory chain is involved in triptolide-induced liver injury, which featured by microvesicular steatosis, hyperlactacidemia and enhanced oxidant stress, although other mechanisms of triptolide-induced liver injury may also exist.

Topics: Animals; Cell Respiration; Chemical and Drug Induced Liver Injury; Diterpenes; Epoxy Compounds; Fatty Liver; Female; Lactic Acid; Liver; Mitochondrial Membranes; Oxidation-Reduction; Oxidative Stress; Phenanthrenes; Plant Extracts; Rats; Rats, Sprague-Dawley; Tripterygium