Juvenile Hormone Diol Kinase

Abstract

Manduca sexta juvenile hormone diol kinase (JHDK) catalyzes the conversion of juvenile hormone (JH) diol to JH diol phosphate. JHDK may be the first example of a phosphotransferase directly involved in the catabolism and inactivation of a lipid-soluble hormone. JHDK is an enzyme crucial for secondary metabolism of JH and possesses high specificity and catalytic efficiency for JH diol. In this study, the purification and characterization of native JHDK are described; its enzymatic properties are examined; and its role in cellular JH metabolism is explored. Using a variety of potential substrates, we show that JHDK has a preference for ATP, but will catalyze the formation of JH diol phosphate with GTP as the phosphate donor. JHDK has a nanomolar K m for JH I diol and a low micromolar value for MgATP. JH II and III diols also serve as phosphate acceptors with low micromolar K m , whereas other diol derivatives of terpenoid esters structurally similar to JH metabolites are not phosphorylated. The reaction proceeds via a sequential Bi Bi mechanism. JHDK is active as a homodimer with a subunit molecular mass of 20 kDa. JHDK binds 5-p-fluorosulfonylbenzoyladenosine and is inhibited by micromolar levels of Ca 2؉ .Juvenile hormones (compound 1; see Fig. 7) are sesquiterpenoids produced in the corpora allata of insects. They travel to target tissues through the hemolymph via a juvenile hormone (JH) 1 -binding protein, where they serve to modulate development, metamorphosis, and sexual maturation (1). During metamorphosis and larval development, JH titers are strictly regulated through biosynthesis and catabolism (2, 3). JH esterase (JHE) and JH epoxide hydrolase are regarded as the enzymes responsible for JH catabolism. However, recent studies have identified a hormonal role for JH acid (compound 2; see Fig. 7) (4, 5), suggesting that JHE is more than an inactivating enzyme. Recently, a cytochrome P450 terpenoid hydroxylase was discovered in the corpora allata of Diploptera punctata; this enzyme may play a role in the temporal suppression of JH titer in the cockroach by metabolizing JH III to 12-hydroxy-JH III (6).Most identifications of JH metabolites in the past have involved studies using either hemolymph or whole organisms, making it difficult to distinguish the role of intracellular JH metabolism. Two studies have been conducted using Manduca sexta tissues dissected and maintained in vitro, one with Malpighian tubules (7) and the other with fat body and imaginal discs (8). The classic model for JH metabolism in insects consists of two primary pathways of inactivation: epoxide hydrolysis to form the diol metabolites and ester cleavage to form the acid metabolites. However, there have been many reports indicating very polar JH metabolites (9 -15). We identified and characterized JH diol phosphate (JHDP; compound 11; Fig. 1), one such polar metabolite, and suggested that it is the principal end product of JH I metabolism in M. sexta (11).We isolated and characterized JH diol kinase (JHDK) from the Malpighian tubules o...

Juvenile Hormone Diol Kinase

“…sexta JHDK Protein Preparations-JHDK was purified through IEF, SAX and WAX chromatography, HIC, and reversed-phase liquid chromatography (RPLC) as described previously (4). * This work was supported by the Nevada Agricultural Experiment Station and was initiated with funding from National Science Foundation Grant IBN-9205637.…”

“…50 g of adult D. melanogaster was ground with a mortar and pestle under liquid N 2 , and the resulting powder was homogenized in 40 ml of ice-cold 10% sucrose containing 5.0 g/ml aprotinin, 0.1 mg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1% mercaptoethanol, and 5.0 g/ml pepstatin A. The following purification protocol for D. melanogaster kinase activity is identical to that described for M. sexta JHDK (4). Briefly, the crude homogenate was centrifuged, and the supernatant was separated by isoelectric focusing (IEF).…”

“…The SCR ␣-helices were constrained by applying a 0.4-kJ penalty to the Ramachandran , angles. 4 An ATP molecule and magnesium were docked to the model structure. The molecules were manually docked using the p21…”

“…These buffers were used, with certain necessary additions, during protein purification and activity measurements. The kinase activity assay and gel chromatography experiments were performed as described in the accompanying article (4).…”

“…Cellular JH metabolism involves a microsomal JH epoxide hydrolase that metabolizes JH to JH diol and a soluble JH diol kinase (JHDK) that catalyzes the formation of JH diol phosphate. JHDK is a homodimer with a subunit molecular mass of 20 kDa that uses MgATP or MgGTP as a phosphate donor to effectively catabolize JH I, II, or III diol by placing a phosphate group on the C-10 hydroxyl, thereby creating JH diol phosphate, the water-soluble end metabolite of JH catabolism (4).…”