Juvenile hormone (JH) esterase: why are you so JH specific?

Abstract

Juvenile hormone esterases (JHEs) from six insects belonging to three orders (Lepidoptera, Coleoptera, and Diptera) were compared in terms of their deduced amino acid sequence and biochemical properties. The four lepidopteran JHEs showed from 52% to 59% identity to each other and about 30% identity to the coleopteran and dipteran JHEs. The JHE of Manduca sexta was remarkably resistant to the addition of organic co-solvents and detergent; in some cases, it demonstrated significant activation of activity. Trifluoromethylketone (TFK) inhibitors with chain lengths of 8, 10 or 12 carbons were highly effective against both lepidopteran and coleopteran JHEs. The coleopteran JHE remained sensitive to TFK inhibitors with a chain length of 6 carbons, whereas the lepidopteran JHEs were significantly less sensitive. When the chain was altered to a phenethyl moiety, the coleopteran JHE remained moderately sensitive, while the lepidopteran JHEs were much less sensitive. The lepidopteran and coleopteran JHEs did not show dramatic differences in specificity to α-naphthyl and ρ-nitrophenyl substrates. However, as the chain length of the α-naphthyl substrates increased from propionate to caprylate, there was a trend towards reduced activity. The JHE of M. sexta was crystallized and the properties of the crystal suggest a high-resolution structure will follow.

Introduction

Using a keen sense of scientific intuition and steady hands for surgical techniques, Wigglesworth, 1935, Wigglesworth, 1936 was the first to identify “juvenile hormone” (JH) as a factor produced by the corpora allata glands that prevented juvenile insects from molting into adults. About 30 years later, Röller et al. (1967) were the first to deduce the chemical structure of juvenile hormone (JH I). Subsequently, Meyer et al. (1970) confirmed the structure of JH I and identified the structure of a second juvenile hormone (JH II). This and other historical aspects of the discovery and characterization of JHs are elegantly reviewed by Gilbert et al. (2000). Perhaps the most wonderful and interesting aspect of JH is the exceptionally diverse range of functionality that JH and/or JH metabolites have on the insect life cycle including roles in development, metamorphosis, reproduction, diapause, migration, polyphenism, and metabolism (reviewed in Roe and Venkatesh, 1990, Riddiford, 1994, de Kort and Granger, 1996, Gilbert et al., 2000). These diverse functionalities suggest that not only are there numerous target sites for JH, but also that its biosynthesis, transport, and degradation must be carefully regulated. Slade and Zibitt (1971) demonstrated that JH was degraded by both ester cleavage and epoxide hydration and that the relative rates of degradation varied with the species and stage of the insect used. As usual, the Gilbert laboratory was one of the first to contribute to the juvenile hormone esterase (JHE) field (Whitmore et al., 1972). Much of the early work on the interaction of hemolymph JH binding proteins and JHE came from the laboratories of John Law and Larry Gilbert (Whitmore and Gilbert, 1972, Kramer et al., 1974, Goodman et al., 1978).

Our laboratory has advanced the hypothesis that regulation of JH is due not only to changes in the rates of its biosynthesis, but also in the rates of its degradation. This has been most clearly shown in lepidopteran larvae where inhibition of JHE reduces the rate of JH degradation and leads to abnormally large larvae and delayed pupation (Sparks and Hammock, 1980, Hammock et al., 1990). Two pathways for the degradation of JH have been intensively studied in insects (reviewed in Hammock, 1985, Roe and Venkatesh, 1990, de Kort and Granger, 1996, Gilbert et al., 2000). One involves the hydrolysis of the methyl ester moiety at one end of the JH molecule by a soluble esterase resulting in the conversion of the methyl ester into a carboxylic acid. The other involves hydrolysis of the epoxide moiety at the other end of the JH molecule by a microsomal epoxide hydrolase resulting in a diol. Both the esterase and hydrolase are members of the α/β-hydrolase fold family and have homologous mechanisms, although they are widely separated in evolutionary history. Our laboratory (Hammock, 1985) has proposed a possible definition of a JH-selective esterase by both biological and biochemical criteria. Biologically, JHE should have an activity that is correlated with a decline in the titer of JH, and the enzyme should be essential for the clearance of JH from the insect’s body. Also, the JHE enzyme should have a low apparent K_m for the substrate JH, and therefore hydrolyze JH with a high k_cat/K_m ratio. Further evidence for supporting the role of an enzyme as a JH-selective esterase could be obtained by specifically reducing (with inhibitors or RNAi) or increasing (by recombinant means or injection) the JH-selective enzyme activity. Of course, we must remember that the term JH-selective esterase may be misleading in that the enzyme may actually be involved in other processes.