Modeling of the bacterial luciferase-flavin mononucleotide complex combining flexible docking with structure-activity data.

Although the crystal structure of Vibrio harveyi luciferase has been elucidated, the binding sites for the flavin mononucleotide and fatty aldehyde substrates are still unknown. The determined location of the phosphate-binding site close to Arg 107 on the alpha subunit of luciferase is supported here by point mutagenesis. This information, together with previous structure-activity data for the length of the linker connecting the phosphate group to the isoalloxazine ring represent important characteristics of the luciferase-bound conformation of the flavin mononucleotide. A model of the luciferase-flavin complex is developed here using flexible docking supplemented by these structural constraints. The location of the phosphate moiety was used as the anchor in a flexible docking procedure performed by conformation search by using the Monte Carlo minimization approach. The resulting databases of energy-ranked feasible conformations of the luciferase complexes with flavin mononucleotide, omega-phosphopentylflavin, omega-phosphobutylflavin, and omega-phosphopropylflavin were filtered according to the structure-activity profile of these analogs. A unique model was sought not only on energetic criteria but also on the geometric requirement that the isoalloxazine ring of the active flavin analogs must assume a common orientation in the luciferase-binding site, an orientation that is also inaccessible to the inactive flavin analog. The resulting model of the bacterial luciferase-flavin mononucleotide complex is consistent with the experimental data available in the literature. Specifically, the isoalloxazine ring of the flavin mononucleotide interacts with the Ala 74-Ala 75 cis-peptide bond as well as with the Cys 106 side chain in the alpha subunit of luciferase. The model of the binary complex reveals a distinct cavity suitable for aldehyde binding adjacent to the isoalloxazine ring and flanked by other key residues (His 44 and Trp 250) implicated in the active site.

Differences in nucleotide specificity and catalytic mechanism between Vibrio harveyi aldehyde dehydrogenase and other members of the aldehyde dehydrogenase superfamily.

The fatty aldehyde dehydrogenase (Vh-ALDH) isolated from the luminescent bacterium, Vibrio harveyi, differs from other aldehyde dehydrogenases in its high affinity for NADP(+). The binding of NADP(+) appears to arise from the interaction of the 2'-phosphate of the adenosine moiety of NADP(+) with a threonine (T175) in the nucleotide recognition site just after the beta(B) strand as well as with an arginine (R210) that pi stacks over the adenosine moiety. The active site of Vh-ALDH contains the usual suspects of a cysteine (C289), two glutamates (E253 and E377) and an asparagine (N147) involved in the aldehyde dehydrogenase mechanism. However, Vh-ALDH has one polar residue in the active site that distinguishes it from other ALDHs; a histidine (H450) is in close contact with the cysteine nucleophile. As a glutamate has been implicated in promoting the nucleophilicity of the active site cysteine residue in ALDHs, the close contact of a histidine with the cysteine nucleophile in Vh-ALDH raises the possibility of alternate routes to increase the reactivity of the cysteine nucleophile. The effects of mutation of these residues on the different functions catalyzed by Vh-ALDH including acylation, (thio)esterase, reductase and dehydrogenase activities should help define the specific roles of the residues in the active site of ALDHs.

A histidine residue in the catalytic mechanism distinguishes Vibrio harveyi aldehyde dehydrogenase from other members of the aldehyde dehydrogenase superfamily.

Aldehyde dehydrogenases (ALDHs) catalyze the transfer to NAD(P) of a hydride ion from a thiohemiacetal derivative of the aldehyde coupled with a cysteine residue in the active site. In Vibrio harveyi aldehyde dehydrogenase (Vh-ALDH), a histidine residue (H450) is in proximity (3.8 A) to the cysteine nucleophile (C289) and is thus capable of increasing its reactivity in sharp contrast to other ALDHs in which more distantly located glutamic acid residues are proposed to act as the general base. Mutation of H450 in Vh-ALDH to Gln and Asn resulted in loss of dehydrogenase, (thio)esterase, and acyl-CoA reductase activities; the residual activity of H450Q was higher than that of the H450N mutant in agreement with the capability of Gln but not Asn to partially replace the epsilon-imino group of H450. Coupled with a change in the rate-limiting step, these results indicate that H450 increases the reactivity of C289. Moreover, for the first time, the acylated enzyme intermediate could be directly monitored after reaction with [(3)H]tetradecanoyl-CoA showing that the H450Q mutant was acylated more rapidly than the H450N mutant. Inactivation of the wild-type enzyme with N-ethylmaleimide was much more rapid than the H450Q mutant which in turn was faster than the H450N mutant, demonstrating directly that the nucleophilicity of C289 was affected by H450. As the glutamic acid residue implicated as the general base in promoting cysteine nucleophilicity in other ALDHs is conserved in Vh-ALDH, elucidation of why a histidine residue has evolved to assist in this function in Vh-ALDH will be important to understand the mechanism of ALDHs in general, as well as help delineate the specific roles of the active site glutamic acid residues.

Hyperactivity and interactions of a chimeric myristoryl-ACP thioesterase from the lux system of luminescent bacteria.

A chimeric myristoyl-ACP thioesterase with much higher catalytic efficiency than the parental enzymes has been generated by ligating the N-terminal half of the lux-specific thioesterase (LuxD) from Photobacterium phosphoreum with the C-terminal half of LuxD from Vibrio harveyi. The LuxD chimera had the same rate-limiting step and specificity, but cleaved esters and thioesters over eight times faster than the native enzymes. LuxD, along with acyl-protein synthetase (LuxE) and reductase (LuxC), comprise a multienzyme complex channeling activated fatty acids into the aldehyde substrate for the bacterial bioluminescence reaction. As P. phosphoreum LuxD and LuxE modulate each of their respective activities, the effects of mixing V. harveyi and the chimeric LuxD with P. phosphoreum LuxE were investigated. The chimeric LuxD stimulated acylation of LuxE to the same extent as V. harveyi LuxD, but to a lower level than that caused by P. phosphoreum LuxD. Conversely, P. phosphoreum LuxE stimulated the thioesterase activity of V. harveyi LuxD by 30% and the chimeric LuxD by 20% while the activity of P. phosphoreum LuxD was increased by over 140%. These results show that the stimulatory effects are unrelated to the level of thioesterase activity and indicate that the carboxyl terminal region of LuxD interacts with LuxE and causes a conformational change.

Mutation of the nucleophilic elbow of the Lux-specific thioesterase from Vibrio harveyi.

Myristoyl-ACP thioesterase (LuxD) from Vibrio harveyi causes the slow release of fatty acids for reduction into the aldehyde substrate required for the bacterial bioluminescence reaction. The active site Ser nucleophile (S(114)) of the LuxD thioesterase is in a gamma-turn with a sequence (AXS(114)XS) quite different from the standard motif of GXSXG found in almost all (thio) esterases and lipases. The presence of an Arg residue (R(118)) in the first turn of the helix after the gamma-turn also distinguishes LuxD from other enzymes. Mutation of R(118) to Leu inactivated the enzyme and prevented acylation of the Ser(114) nucleophile, while even a conservative replacement with Lys resulted in over 75% loss of the same functions, suggesting that R(118) helps maintain the configuration of the active site. In contrast, replacement of S(116) with Gly but not Ala stimulated the esterase and deacylation rates by over threefold. Purification of the S116G mutant to homogeneity and analyses of its intrinsic fluorescence on acylation with myristoyl-CoA clearly demonstrated that this mutant was much more active than wild-type LuxD. The presence of S(116) rather than the expected Gly residue in the gamma-turn containing the Ser nucleophile may function so that release of fatty acids from LuxD is restricted allowing a more efficient delivery of fatty acids to the luminescent system.

Control of luminescence decay and flavin binding by the LuxA carboxyl-terminal regions in chimeric bacterial luciferases.

Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as P. (Vibrio) fischeri, P. phosphoreum, and P. leiognathi, have rapid decay rates. By generation of an X. luminescens-based chimeric luciferase with a 67 amino acid substitution from P. phosphoreum LuxA in the central region of the LuxA subunit, the "slow" X. luminescens luciferase was converted into a chimeric luciferase, LuxA(1)B, with a significantly more rapid decay rate. Two other chimeras with P. phosphoreum sequences substituted closer to the carboxyl terminal of LuxA, LuxA(2)B and LuxA(3)B, retained the characteristic slow decay rates of X. luminescens luciferase but had weaker interactions with both reduced and oxidized flavins, implicating the carboxyl-terminal regions in flavin binding. The dependence of the luminescence decay on concentration and type of fatty aldehyde indicated that the decay rate of "fast" luciferases arose due to a high dissociation constant (K(a)) for aldehyde (A) coupled with the rapid decay of the resultant aldehyde-free complex via a dark pathway. The decay rate of luminescence (k(T)) was related to the decanal concentration by the equation: k(T) = (k(L)A + k(D)K(a))/(K(a) + A), showing that the rate constant for luminescence decay is equal to the decay rate via the dark- (k(D)) and light-emitting (k(L)) pathways at low and high aldehyde concentrations, respectively. These results strongly implicate the central region in LuxA(1)B as critical in differentiating between "slow" and "fast" luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase-flavin-oxygen intermediate.

Change of nucleotide specificity and enhancement of catalytic efficiency in single point mutants of Vibrio harveyi aldehyde dehydrogenase.

The fatty aldehyde dehydrogenase from the luminescent bacterium, Vibrio harveyi (Vh-ALDH), is unique with respect to its high specificity for NADP(+) over NAD(+). By mutation of a single threonine residue (Thr175) immediately downstream of the beta(B) strand in the Rossmann fold, the nucleotide specificity of Vh-ALDH has been changed from NADP(+) to NAD(+). Replacement of Thr175 by a negatively charged residue (Asp or Glu) resulted in an increase in k(cat)/K(m) for NAD(+) relative to that for NADP(+) of up to 5000-fold due to a decrease for NAD(+) and an increase for NADP(+) in their respective Michaelis constants (K(a)). Differential protection by NAD(+) and NADP(+) against thermal inactivation and comparison of the dissociation constants of NMN, 2'-AMP, 2'5'-ADP, and 5'-AMP for these mutants and the wild-type enzyme clearly support the change in nucleotide specificity. Moreover, replacement of Thr175 with polar residues (N, S, or Q) demonstrated that a more efficient NAD(+)-dependent enzyme T175Q could be created without loss of NADP(+)-dependent activity. Analysis of the three-dimensional structure of Vh-ALDH with bound NADP(+) showed that the hydroxyl group of Thr175 forms a hydrogen bond to the 2'-phosphate of NADP(+). Replacement with glutamic acid or glutamine strengthened interactions with NAD(+) and indicated why threonine would be the preferred polar residue at the nucleotide recognition site in NADP(+)-specific aldehyde dehydrogenases. These results have shown that the size and the structure of the residue at the nucleotide recognition site play the key roles in differentiating between NAD(+) and NADP(+) interactions while the presence of a negative charge is responsible for the decrease in interactions with NADP(+) in Vh-ALDH.

Tryptophan fluorescence of the lux-specific Vibrio harveyi acyl-ACP thioesterase and its tryptophan mutants: structural properties and ligand-induced conformational change.

The lux-specific myristoyl-ACP thioesterase from Vibrio harveyi contains four tryptophan residues, Trp23, Trp99, Trp186, and Trp213. Replacement of each of these residues with tyrosine by site-directed mutagenesis coupled with fluorescence and quenching studies of the purified mutant and wild type thioesterases during catalysis has been used to probe ligand-induced conformational changes. Mutant W99Y retained high enzyme activity (80%) with W213Y and W23Y retaining intermediate activity and W186Y having the lowest activity (20%). The sum of the differential fluorescence spectra of the individual tryptophans was identical to the fluorescence spectrum of the wild type thioesterase, showing that mutation had not caused a major conformational change and energy transfer did not occur between the tryptophans. Fluorescence emission maxima and quenching by acrylamide revealed that Trp213 and Trp23 are in a polar environment and/or exposed to solvent while Trp186 appeared to be buried inside the molecule, consistent with the crystal structure of the thioesterase. The fluorescence intensities of the wild type, W23Y, W99Y, and W186Y thioesterases increased in direct correlation to their degree of acylation with myristoyl-CoA, while the fluorescence of the acylated W213Y mutant remained constant, showing that the enhancement of fluorescence was entirely due to interaction of the acyl group with Trp213. Acrylamide quenching of the acylated mutants showed that the accessibility of the tryptophans to solvent was differentially altered and that the quenching of W23Y was enhanced in contrast to the quenching of the other mutants, supporting a ligand-induced conformational change during enzyme turnover.

Conversion of serine-114 to cysteine-114 and the role of the active site nucleophile in acyl transfer by myristoyl-ACP thioesterase from Vibrio harveyi.

The lux-specific myristoyl-ACP thioesterase (LuxD) is responsible for diverting myristic acid into the luminescent system and can function as an esterase and transferase as well as cleave myristoyl-CoA and other thioesters. The recently elucidated crystal structure of the enzyme shows that it belongs to the alpha/beta hydrolase family and that it contains a typical catalytic triad composed of Asp211, His241, and Ser114. What is unusual is that the nucleophilic S114 is not contained within the esterase consensus motif GXSXG although the stereochemistry of the turn involving S114 is almost identical to the nucleophilic elbow found in alpha/beta hydrolases. In contrast to mammalian thioesterases, deacylation of LuxD was the rate-limiting step, with the level of acylated enzyme formed on reaction with myristoyl-CoA and the pre-steady-state burst of p-nitrophenol on cleavage of p-nitrophenyl myristate both being 0.7 mol/mol. Cold chase experiments showed that the deacylation rate of LuxD corresponded closely to the turnover rate of the enzyme with ester or thioester substrates. Replacement of S114 by a cysteine residue generated a mutant (S114C) that was acylated with the same pH dependence as LuxD but had greatly diminished capacity to transfer acyl groups to water or glycerol. The acyl group could be removed from the S114C mutant by neutral hydroxylamine, showing that a cysteine residue had been acylated. Mutation of H241 creating the double mutant, S114C.H241N, decreased acylation of the cysteine residue. These results provide direct kinetic and chemical evidence that S114 is the site of acylation linked to H241 in the charge relay system and have led to the recognition of a more general consensus motif flanking the nucleophilic serine in thioesterases.

The role of lux autoinducer in regulating luminescence in Vibrio harveyi; control of luxR expression.

Analysis of Vibrio harveyi dark autoinducer mutants has demonstrated that the level of LuxR was much lower than that observed in wild-type cells. Complementation with luxR fully restored luminescence suggesting that the lux autoinducer may control expression of the luxR regulatory gene. By primer extension, the transcriptional start site of luxR was located 78 bp from the initiation codon. The level of the primer-extended product was enhanced upon addition of the lux autoinducer to the autoinducer mutants, which was confirmed by hybridization of lux mRNA with specific probes. By using chloramphenicol acetyltransferase as a reporter gene in a transcriptional fusion with luxR, the stimulatory effect of autoinducer on luxR expression was shown to occur at the level of the luxR promoter. The results provide evidence that the autoinducer signal in V. harveyi can be transduced through luxR, resulting in stimulation of luminescence.

Involvement of cysteine 289 in the catalytic activity of an NADP(+)-specific fatty aldehyde dehydrogenase from Vibrio harveyi.

Fatty aldehyde dehydrogenase (Vh.ALDH) from the luminescent bacterium, Vibrio harveyi, may be implicated in controlling luminescence as it catalyzes the oxidation of the fatty aldehyde substrate for the light-emitting reaction. On the basis of the amino-terminal sequence of Vh.ALDH, a degenerate probe was used to screen a genomic library of V harveyi in pBR322, a positive clone was selected containing the Vh.ALDH gene and expressed in Escherichia coli, and the enzyme was purified to homogeneity. Although the sequence of the V. harveyi ALDH significantly diverged from other aldehyde dehydrogenases, mutation of a conserved cysteine implicated in catalysis completely inactivated the enzyme without loss of its ability to bind nucleotides, consistent with a catalytic role for this residue. Using absorption and fluorescence assays for NAD(P)H, it was shown that NAD+ and NADP+ bound to the same site and that saturation of Vh.ALDH with NADP+ occurred with a Michaelis constant (Km = 1.4 microM) over 40 times lower than that reported for other aldehyde dehydrogenases. Although V. harveyi aldehyde dehydrogenase is unique in terms of its high specificity for NADP+, the identification of a catalytic conserved cysteine in Vh.ALDH clearly indicates that a highly related mechanism and structure have been retained among even the most diverged aldehyde dehydrogenases.

Structure of a myristoyl-ACP-specific thioesterase from Vibrio harveyi.

The crystal structure of a myristoyl acyl carrier protein specific thioesterase (C14ACP-TE) from a bioluminescent bacterium, Vibrio harveyi, was solved by multiple isomorphous replacement methods and refined to an R factor of 22% at 2.1-A resolution. This is the first elucidation of a three-dimensional structure of a thioesterase. The overall tertiary architecture of the enzyme resembles closely the consensus fold of the rapidly expanding superfamily of alpha/beta hydrolases, although there is no detectable homology with any of its members at the amino acid sequence level. Particularly striking similarity exists between the C14ACP-TE structure and that of haloalkane dehalogenase from Xanthobacter autotrophicus. Contrary to the conclusions of earlier studies [Ferri, S. R., & Meighen, E. A. (1991) J. Biol. Chem. 266, 12852-12857] which implicated Ser77 in catalysis, the crystal structure of C14ACP-TE reveals a lipase-like catalytic triad made up of Ser114, His241, and Asp211. Surprisingly, the gamma-turn with Ser114 in a strained secondary conformation (phi = 53 degrees, psi = -127 degrees), characteristic of the so-called nucleophilic elbow, does not conform to the frequently invoked lipase/esterase consensus sequence (Gly-X-Ser-X-Gly), as the positions of both glycines are occupied by larger amino acids. Site-directed mutagenesis and radioactive labeling support the catalytic function of Ser114. Crystallographic analysis of the Ser77-->Gly mutant at 2.5-A resolution revealed no structural changes; in both cases the loop containing the residue in position 77 is disordered.(ABSTRACT TRUNCATED AT 250 WORDS)

Subunit interactions and the role of the luxA polypeptide in controlling thermal stability and catalytic properties in recombinant luciferase hybrids.

Bacterial luciferases with over 70% sequence identity from the terrestrial species, Xenorhabdus luminescens, and the marine species, Vibrio harveyi, exhibit large differences in thermal stability (Szittner and Meighen, 1990, J. Biol. Chem. 265, 16581-16587). The origin of these differences was investigated with genetically constructed hybrids containing one subunit from X. luminescens and the other from V. harveyi. While no activity was detected with the single (alpha and beta) subunits both in vitro and in vivo, the recombinant hybrid luciferases (alpha Xl beta Vh and alpha Vh beta Xh) were highly active and could be purified to homogeneity. The kinetic properties of the hybrid enzymes including aldehyde specificity, flavin binding and luminescence decay rates, were found to be nearly identical to those of the native luciferases (alpha Xl beta Xl or alpha Vh beta Vh) containing the same alpha subunit. In addition, the rate of thermal inactivation and temperature dependent quenching of the intrinsic fluorescence by flavin were found to be independent of the nature of the beta subunit, quite opposite to previous reports that the thermal stability is controlled by the beta subunit. Thus, the alpha subunit appears primarily responsible for controlling both the catalytic and structural properties of luciferase.

The gene convergent to luxG in Vibrio fischeri codes for a protein related in sequence to RibG and deoxycytidylate deaminase.

The nucleotide sequence of a convergent gene with the same bidirectional transcriptional terminator as the Vibrio fischeri lux operon has been determined. This gene codes for a polypeptide of 147 amino acids which is related in sequence to the polypeptide coded by the first gene (ribG) of the rib operon of Bacillus subtilis as well as deoxycytidylate deaminase of T4 bacteriophage and Saccharomyces cerevisiae. These results raise the possibility of a linkage between the regulation of the lux genes and riboflavin synthesis in Vibrio fischeri.

Multiple repetitive elements and organization of the lux operons of luminescent terrestrial bacteria.

The complete nucleotide sequences of the luxA to luxE genes, as well as the flanking regions, were determined for the lux operons of two Xenorhabdus luminescens strains isolated from insects and humans. The nucleotide sequences of the corresponding lux genes (luxCDABE) were 85 to 90% identical but completely diverged 350 bp upstream of the first lux gene (luxC) and immediately downstream of the last lux gene (luxE). These results show that the luxG gene found immediately downstream of luxE in luminescent marine bacteria is missing at this location in terrestrial bacteria and raise the possibility that the lux operons are at different positions in the genomes of the X. luminescens strains. Four enteric repetitive intergenic consensus (ERIC) or intergenic repetitive unit (IRU) sequences of 126 bp were identified in the 7.7-kbp DNA fragment from the X.luminescens strain isolated from humans, providing the first example of multiple ERIC structures in the same operon including two ERIC structures at the same site. Only a single ERIC structure between luxB and luxE is present in the 7-kbp lux DNA from insects. Analysis of the genomic DNAs from five X. luminescens strains or isolates by polymerase chain reaction has demonstrated that an ERIC structure is between luxB and luxE in all of the strains, whereas only the strains isolated from humans had an ERIC structure between luxD and luxA. The results indicate that there has been insertion and/or deletion of multiple 126-bp repetitive elements in the lux operons of X.luminescens during evolution.

The lux genes of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish. Nucleotide sequence, difference in gene organization, and high expression in mutant Escherichia coli.

The lux genes required for light expression in the luminescent bacterium Photobacterium leiognathi (ATCC 25521) have been cloned and expressed in Escherichia coli and their organization and nucleotide sequence determined. Transformation of a recombinant 9.5-kbp chromosomal DNA fragment of P. leiognathi into an E. coli mutant (43R) gave luminescent colonies that were as bright as those of the parental strain. Moreover, expression of the lux genes in the mutant E. coli was strong enough so that not only were high levels of luciferase detected in crude extracts, but the fatty-acid reductase activity responsible for synthesis of the aldehyde substrate for the luminescent reaction could readily be measured. Determination of the 7.3-kbp nucleotide sequence of P. leiognathi DNA, including the genes for luciferase (luxAB) and fatty-acid reductase (luxCDE) as well as a new lux gene (luxG) found recently in luminescent Vibrio species, showed that the order of the lux genes was luxCDABEG. Moreover, luxF, a gene homologous to luxB and located between luxB and luxE in Photobacterium but not Vibrio strains, was absent. In spite of this different lux gene organization, an intergenic stem-loop structure between luxB and luxE was discovered to be highly conserved in other Photobacterium species after luxF.

Structure and properties of luciferase from Photobacterium phosphoreum.

The nucleotide sequences of the luxA and luxB genes coding for the alpha and beta subunits, respectively, of luciferase from Photobacterium phosphoreum have been determined. The predicted amino acid sequences of the alpha and beta subunits were shown to be significantly different from other bacterial luciferases with 62 to 88% identity with the alpha subunits and 47 to 71% identity with the beta subunits of other species. Expression of the different luciferases appear to correlate with the number of modulator codons. Kinetic properties of P. phosphoreum luciferase were shown to reflect the bacterium's natural cold temperature habitat.

Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium.

The lux genes required for expression of luminescence have been cloned from a terrestrial bacterium, Xenorhabdus luminescens, and the nucleotide sequences of the luxA and luxB genes coding for the alpha and beta subunits of luciferase determined. The lux gene organization was closely related to that of marine bacteria from the Vibrio genus with the luxD gene being located immediately upstream and the luxE downstream of the luciferase genes, luxAB. A high degree of homology (85% identity) was found between the amino acid sequences of the alpha subunits of X. luminescens luciferase and the luciferase from a marine bacterium, Vibrio harveyi, whereas the beta subunits of the two luciferases had only 60% identity in amino acid sequence. The similarity in the sequences of the alpha subunits of the two luciferases was also reflected in the substrate specificities and turnover rates with different fatty aldehydes supporting the proposal that the alpha subunit almost exclusively controls these properties. The luciferase from X. luminescens was shown to have a remarkably high thermal stability being stable at 45 degrees C (t 1/2 greater than 3 h) whereas V. harveyi luciferase was rapidly inactivated at this temperature (t 1/2 = 5 min). These results indicate that the X. luminescens lux system may be the bacterial bioluminescent system of choice for application in coupled luminescent assays and expression of lux genes in eukaryotic systems at higher temperatures.

Determination of the release rate of aldehyde pheromones from insect lures by cold trapping and direct bioluminescence analysis.

A rapid and sensitive procedure for measuring the release rate of aldehyde pheromones from insect lures has been developed. Using cold traps to condense aldehydes released into an airstream, the amount of pheromone in the aqueous condensate could be directly analyzed using a luminescence assay based on the response of bacterial luciferase to long chain aldehydes. A high recovery (approximately 70%) of aldehyde pheromone in the cold trap was obtained even when amounts as low as 10 ng were released into the airstream. Trapping times as low as 5 min can be used and analysis requires only a few seconds after temperature equilibration of the sample. This approach was applied to measure the release rate of 11-tetradecenal from different spruce budworm lures as well as to demonstrate that the release of aldehyde from a lure containing [14C]-cis-9-hexadecenal correlated closely to the release rate of radioactive material.

Development of a bioluminescence assay for aldehyde pheromones of insects : II. Analysis of pheromone glands.

Pheromone levels in the glands of individual female moths of the spruce budworm (Choristoneura fumiferana), the western spruce budworm (C. occidentalis), the navel orangeworm (Amyelois transitella), and the corn earworm (Heliothis zea) were quantitively measured by means of a new bacterial bioluminescence assay specific for aldehydes. The sensitivity and rapidity of the bioluminescent assay enabled studies to be conducted on the dependence of the pheromone levels in the spruce budworm on age and the effect of photoperiod on the pheromone levels in the corn earworm. The bioluminescence assay provides a rapid and sensitive approach for studying aldehyde pheromone levels and their regulation in insects.