Rose Szittner

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 α 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, ω‐phosphopentylflavin, ω‐phosphobutylflavin, and ω‐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 α 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.

Rate of pheromone release by individual spruce budworm moths

Abstract The rate and temporal pattern of pheromone emission by single and grouped female spruce budworm moths were measured by combining the trapping of pheromone on Porapak Q with a rapid luciferase bioassay developed for aldehyde pheromone. Pheromone release occurred mainly during the scotophase of a 12:12 L:D cycle in a series of bursts (up to 50 ng/hr) with considerable variability observed between insects. Analysis of over 30 individual females showed that 30% release no detectable pheromone, 60% release between 20 and 100 ng of pheromone and 10% release greater than 100 ng in a 24-hr period. Overall, a single female (1–3 days old) released an average of 60 ± 50 ng (± S.D.) of pheromone per night with a total of 260 ± 210 ng (± S.D.) being released over its life span (∼7 days). Grouped females released lower quantities of pheromone. The amount of pheromone in the glands of female moths also displayed a rhythm with the levels beings higher later in the day than at the start of the photophase.

The role of the 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.

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.

Bright stable luminescent yeast using bacterial luciferase as a sensor.

Bright luminescent yeast cells with light intensities similar to bacteria containing luciferase (LuxAB) were generated by providing saturating nontoxic levels of the substrates for the bioluminescence reaction (FMNH(2)+O(2) and fatty aldehyde-->light). Z-9-Tetradecenal added to yeast (+luxAB) gave a luminescent signal close to that with decanal with the signal remaining strong for >24h while luminescence of yeast with decanal decayed to less than 0.01% of that with Z-9-tetradecenal after 2min. Moreover, yeast survived in 0.5% (v/v) Z-9-tetradecenal while 0.005% (v/v) decanal was lethal. Luminescence of yeast (+luxAB) was also stimulated 100-fold by transformation with the NADPH-specific FMN reductase (FRP) from Vibrio harveyi. The recognition of the nontoxicity and high luminescence generated by Z-9-tetradecenal and the generation of high levels of FMNH(2) in yeast by transformation with a flavin reductase provide evidence for the strong potential use of bacterial luciferase as the light-emitting sensor of choice in eukaryotic organisms.

Development of a bioluminescence assay for aldehyde pheromones of insects

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.

Adsorptive immobilization of bacterial luciferases on alkyl-substituted Sepharose 4B

Abstract Alkyl-substituted Sepharose 4B prepared by the glycidyl ether method with different degrees of substitution and alkyl chain length, have been used as a non-ionic matrix for immobilization of bacterial luciferases. Exposure of hydrophobic clusters in the protein molecule was confirmed by fluorescence studies using 8-anilino-1-napthalene-sulfonate as a hydrophobic reporter. Immobilization of luciferases took place with high efficiency and retention of their basic kinetic properties including a high recovery of activity, which was dependent on the α and not the β subunit of the heterodimeric enzyme. A remarkable increase in thermal stability was shown for Vibrio harveyi (Vh) luciferase on binding to octyl-Sepharose. As FMN reductase was coimmobilized with Vh luciferase, the reducing power for the reaction (FMNH 2 ) could also be supplied by NAD(P)H. The catalytic potential of the coimmobilized enzymes was dramatically improved apparently due to reduction of FMNH 2 auto-oxidation, as reported earlier. The potential application of this method to measure different compounds that can reduce NAD(P) is thereby increased. The simplicity of adsorption of luciferase on these matrices together with the high activity, reusability and increased thermal stability suggests that the method of immobilization described may provide a useful procedure for bioassays when coupled to the bacterial luminescence reaction.

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 bacteriums natural cold temperature habitat.

Development of a bioluminescence assay for aldehyde pheromones of insects : III. Analysis of airborne pheromone.

A newly developed bioluminescent assay was used to measure quantitatively the amount of (E)-11-tetradecenal, the major component of the sex pheromone of the spruce budworm, trapped on Porapak Q®. The bioluminescent response was linearly related to the amount of aldehyde either deposited on the absorbent or trapped from an airstream. However, the recovery of pheromone from Porapak was dependent on whether the air was prefiltered (through Porapak) or taken directly from the atmosphere. Furthermore, pheromone on Porapak was lost with time during the flow of air through the absorbent, indicating that trapping of aldehyde pheromone should be conducted for short periods of time for optimal recoveries. The applicability of the assay system for the rapid and direct measurement of the release rates of aldehyde pheromone lures was demonstrated for pheromone lures used for baiting spruce budworm traps.

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.