Hubert Wojtasek

Conformational change in the pheromone-binding protein from Bombyx mori induced by pH and by interaction with membranes.

The pheromone-binding protein (PBP) fromBombyx mori was expressed in Escherichia coliperiplasm. It specifically bound radiolabeled bombykol, the natural pheromone for this species. It appeared as a single band both in native and SDS-polyacrylamide gel electrophoresis and was also homogeneous in most chromatographic systems. However, in ion-exchange chromatography, multiple forms sometimes appeared. Attempts to separate them revealed that they could be converted into one another. Analysis of the protein by circular dichroism and fluorescence spectroscopy demonstrated that its tertiary structure was sensitive to pH changes and that a dramatic conformational transition occurred between pH 6.0 and 5.0. This high sensitivity to pH contrasted markedly with its thermal stability and resistance to denaturation by urea. There was also no significant change in CD spectra in the presence of the pheromone. The native protein isolated from male antennae displayed the same changes in its spectroscopic properties as the recombinant material, demonstrating that this phenomenon is not an artifact arising from the expression system. This conformational transition was reproduced by interaction of the protein with anionic (but not neutral) phospholipid vesicles. Unfolding of the PBP structure triggered by membranes suggests a plausible mechanism for ligand release upon interaction of the PBP-pheromone complex with the surface of olfactory neurons. This pH-linked structural flexibility also explains the heterogeneity reported previously for B. mori PBP and other members of this class of proteins.

Degradation of an alkaloid pheromone from the pale-brown chafer, Phyllopertha diversa (Coleoptera: Scarabaeidae), by an insect olfactory cytochrome P450

The pale‐brown chafer, Phyllopertha diversa, utilizes an unusual alkaloid, 1,3‐dimethyl‐2,4‐(1H,3H)‐quinazolinedione, as its sex pheromone. This compound is rapidly degraded in vitro by the antennal protein extracts from this scarab beetle. Demethylation at the N‐1 position and hydroxylation of the aromatic ring have been identified as the major catabolic pathways. The enzyme responsible for the pheromone degradation is membrane‐bound, requires NAD(P)H for activity and is sensitive to cytochrome P450 inhibitors, such as proadifen and metyrapone. The ability to metabolize this unusual pheromone was not detected in 12 species tested, indicating that the P450 system, specific to male P. diversa antennae, has evolved as a mechanism for olfactory signal inactivation.

Biochemistry of Proteins That Bind and Metabolize Juvenile Hormones.

A diverse group of proteins has evolved to bind and metabolize insect juvenile hormones (JHs). Synthetic radiolabeled JHs and their photoaffinity analogs have enabled us to isolate and characterize JH binding proteins (JHBPs), a putative nuclear JH receptor, JH esterases (JHEs), JH epoxide hydrolases (JHEHs), and methyl farnesoate binding proteins (MFBPs). Highlights of recent progress on structural characterization of JHBPs and JHEHs of two lepidopterans will be described. Efforts to identify MFBPs of penaeid shrimp will be discussed, and the discovery of a possible vertebrate JHBP will be presented.

In vitro modeling of the ternary interaction in juvenile hormone metabolism

The gradual decline in juvenile hormone (JH) titer followed by its complete clearance early in the last larval instar is required for the onset of the metamorphosis of lepidopterous larvae. JH titer is regulated by both biosynthesis and degradation. Two major pathways for JH metabolism, ester hydrolysis and epoxide hydration, are due to JH esterase (JHE) and JH epoxide hydrolase (JHEH), respectively. In vitro experiments designed to elucidate the molecular mechanism of JH metabolism are described. First, microsomal JHEH in Manduca sexta eggs was identified by using photoaffinity analogs of JH, and purified to homogeneity with ion exchange and hydroxylapatite columns. Purified JHEH from M. sexta eggs was kinetically characterized. The effects of pH and various reagents imply that JHEH in M. sexta eggs and mammalian microsomal EH strongly resemble each other. JH binding protein (JHBP) appears to protect JH from JHEH; however, the hydration of JH-acid is not affected by JHBP because JH-acid is not bound by JHBP, but is a good substrate for JHEH. Thus, the major function of JHEH in M. sexta eggs, which has a cytosolic JHBP, is likely to act as the ultimate scavenger for JH by hydrating JH-acid. Second, although virtually all JH exists as the JHBP·JH complex in hemolymph, JHE nonetheless effectively hydrolyzes JH in a JHBP·JH complex. Circular dichroism experiments suggest the possibility of a direct interaction between JHBP and JHE. These experimental approaches using reconstituted in vitro model systems may elucidate some of the complex interactions in the JH signaling and metabolic pathways.

Perireceptor Events in Pheromone Perception in Scarab Beetles

Abstract Despite the remarkable diversity of the sex pheromone chemistry in scarab beetles, various species utilize a common type of γ-lactones in their chemical communication channels. These compounds differ primarily in length of the alkenyl side chain and the stereochemistry at the chiral center. Two species, Anomala osakana and Popillia japonica , utilize the opposite enantiomers of japonilure as sex pheromones. Each species produces only one of the enantiomers that functions as its own sex pheromone and as a behavioral antagonist to the alloreceiver. Pheromone binding proteins (PBPs) have been characterized, which are present in these and several other scarab species. In most cases there was only one class of PBP, which was expressed in both sexes. A. osakana and P. japonica possess each one single PBP with high homology to each other. In each species the same PBP seems to recognize both enantiomers of japonilure, i. e., the pheromonal and the “stop” signals. Based on the N-terminal sequences, the antennae-specific proteins from various other species were highly conserved within the family and showed moderate homology to putative odorant binding protein from Drosophila melanogaster (47%), Lygus lineolaris (45∼50%) and the ABPX protein from Bombyx mori (30∼35%). From analysis of extracts of soluble antennal proteins from several species, significant degradation of the γ-lactones (buibuilactone, japonilure) was detected, essentially in all of them, even in species that do not use these compounds as pheromones. Recently a peculiar pheromone with a diamide moiety [1, 3-dimethyl-2, 4-(1 H , 3 H )-quinazolinedione] was isolated from Phyllopertha diversa , which was rapidly degraded by antennal enzymes from this species. Beetles that utilize lactones as their pheromones possess little or no ability to metabolize this compound.

Phosphonic Acid Analogues of Tyrosine and Dihydroxyphenylalanne (DOPA) as Tyrosinase Inhibitors

Structural differences between carboxylic and phosphonic acid groups do not prevent aminophosphonic acids from serving as substrates of some enzymes that normally utilize amino acids. This is not particularly surprising if considering the fact that most of these enzymes catalyze reactions taking place without direct involvement of carboxylic moiety. A good example is the interaction of phosphonic analogues of tyrosine with tyrosinase. A simple replacement of carboxylic acid group by phosphonic acid moiety led to compound 1 which serves as good synthetic substrate of tyrosinase.1 Homologues of tyrosine exert, however, quite surprising properties. Compound 2 possessing additional methylene group in a side chain also appeared to be a substrate, whereas compound 3, obtained by shortening of the alkyl chain of 1, turned out to be quite powerful inhibitor of the enzyme.2 All of these compounds form complexes with tyrosinase by a fit of the aromatic part of the molecule into catechol-binding site of the enzyme and by electrostatic complexation of negatively charged dianion by the positively charged, carboxylate-binding portion of the enzyme.