No abstract

The sesquiterpenoid juvenile hormone (JH) regulates insect development and reproduction. Most insects produce only one chemical form of JH, but the Lepidoptera produce four derivatives featuring ethyl branches. The biogenesis of these JHs requires the synthesis of ethyl-substituted farnesyl diphosphate (FPP) by FPP synthase (FPPS). To determine if there exist more than one lepidopteran FPPS, and whether one FPPS homolog is better adapted for binding the bulkier ethyl-branched substrates/products, we cloned three lepidopteran FPPS cDNAs, two from Choristoneura fumiferana and one from Pseudaletia unipuncta. Amino acid sequence comparisons among these and other eukaryotic FPPSs led to the recognition of two lepidopteran FPPS types. Type-I FPPSs display unique active site substitutions, including several in and near the first aspartate-rich motif, whereas type-II proteins have a more "conventional" catalytic cavity. In a yeast assay, a Drosophila FPPS clone provided full complementation of an FPPS mutation, but lepidopteran FPPS clones of either type yielded only partial complementation, suggesting unusual catalytic features and/or requirements of these enzymes. Although a structural analysis of lepidopteran FPPS active sites suggested that type-I enzymes are better suited than type-II for generating ethyl-substituted products, a quantitative real-time PCR assessment of their relative abundance in insect tissues indicated that type-I expression is ubiquitous whereas that of type-II is essentially confined to the JH-producing glands, where its transcripts are approximately 20 times more abundant than those of type-I. These results suggest that type-II FPPS plays a leading role in lepidopteran JH biosynthesis in spite of its apparently more conventional catalytic cavity.

In steroid biosynthesis, human dehydroepiandrosterone sulfotransferase (DHEA-ST) in the adrenals has been reported to catalyze the transfer of the sulfonate group from 3-phosphoadenosine-5-phosphosulfate to dehydroepiandrosterone (DHEA). DHEA and its sulfate play roles as steroid precursors; however, the role of the enzyme in the catabolism of androgens is poorly understood. Androsterone sulfate is clinically recognized as one of the major androgen metabolites found in urine. Here it is demonstrated that this enzyme recognizes androsterone (ADT) as a cognate substrate with similar kinetics but a 2-fold specificity and stronger substrate inhibition than DHEA. The structure of human DHEA-ST in complex with ADT has been solved at 2.7 Å resolution, confirming ADT recognition. Structural analysis has revealed the binding mode of ADT differs from that of DHEA, despite the similarity of the overall structure between the ADT and the DHEA binary complexes. Our results identify that this human enzyme is an ADT sulfotransferase as well as a DHEA sulfotransferase, implying an important role in steroid homeostasis for the adrenals and liver.Sulfonation is catalyzed by a family of sulfotransferases that conjugate a sulfonate group (SO 3 ) from 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS) 1 to a hydroxyl group of the recipient molecule. With desulfation by sulfatases, sulfonation has been considered as one of the major enzymatic reactions in the metabolism not only of endogenous compounds and xenobiotics, but also of steroid hormones. In most cases, the transfer of the charged sulfonate moiety to an acceptor steroid decreases the biological activity of the steroid. Indeed, steroid sulfates resulting from this reaction are not capable of binding to or activating steroid receptors. In addition, the sulfonation reaction increases water solubility of steroids and thereby enhances their excretion into the urine and/or bile (1, 2).Human dehydroepiandrosterone sulfotransferase (DHEA-ST; SULT2A1; EC 2.8.2.2) was identified mainly from human liver and adrenals, using Northern blot analysis (3) and RT-PCR analysis (4). A single isoform of DHEA-ST from human liver and adrenal tissues was confirmed by the expression and purification of the enzyme from these organs (5, 6), molecular cloning studies (7) and the comparison study of the physical, kinetic, and immunological properties of liver and adrenal forms of the enzyme (8). Steroid sulfonation has been recognized as an important means for maintaining steroid hormone levels in their metabolism. In humans, dehydroepiandrosterone sulfate (DHEAS) is the most prodigious steroid precursor and one of the major secretory products of both adult and fetal adrenals. In the fetoplacental-maternal unit (the unique interdependence of fetus, placenta, and mother) shown in Scheme 1, DHEAS plays an important role as the major precursor for placental estrogen biosynthesis, thus maintaining pregnancy. A considerable amount of DHEAS is mainly produced from the fetal zone in the adrenal gland (9). Then DHEAS...

Phosphorylation-dependent protein-protein interaction has rarely been targeted in medicinal chemistry. Thymoquinone, a naturally occurring antitumor agent, disrupts prephosphorylated substrate recognition by the polo-box domain of polo-like kinase 1, a key mitotic regulator responsible for various carcinogenesis when overexpressed. Here, crystallographic studies reveal that the phosphoserine/phosphothreonine recognition site of the polo-box domain is the binding pocket for thymoquinone and its analogue poloxime. Both small molecules displace phosphopeptides bound with the polo-box domain in a slow but noncovalent binding mode. A conserved water bridge and a cation-π interaction were found as their competition strategy against the phosphate group. This mechanism sheds light on small-molecule intervention of phospho-recognition by the polo-box domain of polo-like kinase 1 and other phospho-binding proteins in general.