Cysteine-286 as the site of acylation of the Lux-specific fatty acyl-CoA reductase.

The channelling of fatty acids into the fatty aldehyde substrate for the bacterial bioluminescence reaction is catalyzed by a fatty acid reductase multienzyme complex, which channels fatty acids through the thioesterase (LuxD), synthetase (LuxE) and reductase (LuxC) components. Although all three components can be readily acylated in extracts of different luminescent bacteria, this complex has been successfully purified only from Photobacterium phosphoreum and the sites of acylation identified on LuxD and LuxE. To identify the acylation site on LuxC, the nucleotide sequence of P. phosphoreum luxC has been determined and the gene expressed in a mutant Escherichia coli strain. Even in crude extracts, the acylated reductase intermediate as well as acyl-CoA reductase activity could be readily detected, providing the basis for analysis of mutant reductases. Comparison of the amino-acid sequences of LuxC from P. phosphoreum, P. leiognathi and other luminescent bacteria, showed that only three cysteine residues (C171, C279, and C286) were conserved. As a cysteine residue on LuxC has been implicated in fatty acyl transfer, each of the conserved cysteine residues of the P. phosphoreum and P. leiognathi reductases was converted to a serine residue, and the properties of the mutant proteins examined. Only mutation of C286-blocked reductase activity and prevented formation of the acylated reductase intermediate, showing that C286 is the site of acylation on LuxC.

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.

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.

The LuxR regulator protein controls synthesis of polyhydroxybutyrate in Vibrio harveyi.

The LuxR regulatory protein of Vibrio harveyi has been shown to control synthesis of polyhydroxybutyrate (PHB) as well as luminescence so as to occur at high cell density, suggesting that it is a general regulatory protein. Mutants defective in the production of LuxR (D1, D34, and MR1130) were found to be missing PHB, whose synthesis could be restored by complementation with luxR. Triparental mating with a V. harveyi genomic library revealed the presence of three genomic clones (G1, G2 and G3) that could also restore PHB synthesis and luminescence to cells which express low levels of luxR (D1 and D34) but not to luxR- cells (MR1130) suggesting that luxR expression was being stimulated. Analyses of luxR mRNA levels by mRNA dot blot hybridization and by primer extension confirmed that luxR mRNA levels were increased 4 to 7-fold in the D1 and D34 cells by the G1, G2 and G3 fragments and show that expression of a single genomic copy of luxR is sufficient to restore synthesis of PHB. The results demonstrate that V. harveyi LuxR controls the induction of a process not intimately involved in the bioluminescence system and clearly distinguishes its role in V. harveyi from that of LuxR from Vibrio (Photobacterium) fischeri, which has only been associated with regulation of light emission.

Cloning and functional studies of a luxO regulator LuxT from Vibrio harveyi.

LuxO is the central regulator integrating the quorum sensing signals controlling autoinduction of luminescence in Vibrio harveyi. We have previously purified to homogeneity a new lux regulator, LuxT, that binds to the luxO promoter. Based on the sequence of the tryptic peptides of LuxT, degenerate oligonucleotides were designed for PCR of the genomic DNA. A 273 bp PCR DNA fragment containing sequences encoding the tryptic peptides was extended by inverse PCR to obtain the complete gene (luxT) encoding a protein of 153 amino acids which shares homology with the AcrR/TetR family of transcriptional regulators. The recombinant and native LuxT gave the same footprint binding between 117 and 149 bp upstream from the luxO initiation codon. Gene disruption of luxT in V. harveyi increased luxO expression and affected the cell density dependent induction of luminescence showing that LuxT was a repressor of luxO. As LuxT also affected the survival of the V. harveyi cells at high salt concentration and homologous proteins are present in other bacterial species, including the pathogen, Vibrio cholerae, the LuxT regulatory protein appears to be a general rather than a lux-specific regulator.

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.

Identification of the acyl transfer site of fatty acyl-protein synthetase from bioluminescent bacteria.

Fatty acid activation, transfer, and reduction by the fatty acid reductase multienzyme complex from Photobacterium phosphoreum to generate fatty aldehydes for the luminescence reaction is regulated by the interaction of the synthetase and reductase subunits of this complex. Identification of the specific site involved in covalent transfer of the fatty acyl group between the sites of activation and reduction on the synthetase and reductase subunits, respectively, is a critical step in understanding how subunit interactions modulate the flow of fatty acyl groups through the fatty acid reductase complex. To accomplish this goal, the nucleotide sequence of the luxE gene coding for the acyl-protein synthetase subunit (373 amino acid residues) was determined and the conserved cysteinyl residues implicated in fatty acyl transfer identified. Using site-specific mutagenesis, each of the five conserved cysteine residues was converted to a serine residue, the mutated synthetases expressed in Escherichia coli, and the properties of the mutant proteins examined. On complementation of four of the mutants with the reductase subunit, the synthetase subunit was acylated and the acyl group could be reversibly transferred between the reductase and synthetase subunits, and fatty acid reductase activity was fully regenerated. As well, sensitivity of the acylated synthetases to hydroxylamine cleavage (under denaturation conditions to remove any conformational effects on reactivity) was retained, showing that a cysteine and not a serine residue was still acylated. However, substitution of a cysteine residue only ten amino acid residues from the carboxyl terminal (C364S) prevented acylation of the synthetase and regeneration of fatty acid reductase activity. Moreover, this mutant protein preserved its ability to activate fatty acid to fatty acyl-AMP but could not accept the acyl group from the reductase subunit, demonstrating that the C364S synthetase had retained its conformation and specifically lost the fatty acylation site. These results provide evidence that the flow of fatty acyl groups in the fatty acid reductase complex is modulated by interaction of the reductase subunit with a cysteine residue very close to the carboxyl terminal of the synthetase, which in turn acts as a flexible arm to transfer acyl groups between the sites of activation and reduction.

Expression, purification and crystallization of the Vibrio harveyi acyltransferase.

We have obtained X-ray quality single crystals of Vibrio harveyi acyltransferase. The protein was obtained from V. harveyi by a gene mobilization expression system. The crystals are monoclinic (space group P2(1), a = 89.9 A, b = 83.6 A, c = 47.1 A, beta = 97.3 degrees) with two molecules related by a pronounced non-crystallographic dyad in the asymmetric unit, with a solvent content of approximately 50%. The diffraction pattern from fresh crystals extends beyond 2 A resolution using sealed tube CuK alpha radiation. The elucidation of the three-dimensional structure of this enzyme, believed to contain a proteinase-like catalytic triad, which resembles in many ways other eukaryotic fatty acid chain terminating enzymes, may have important consequences for our understanding of the molecular basis of the final stages of the synthesis of fatty acids.

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.

A miniaturized Ames mutagenicity assay employing bioluminescent strains of Salmonella typhimurium.

Increased awareness of the role of environmental factors in carcinogenesis has led to an emphasis on preventing or minimizing exposure to genotoxicants. This is presently promoting the development of simple, rapid, cost-effective mutagenicity screening assays. We have developed a test system based on the well-known Salmonella mutagenicity assay. The lux genes, which permit cells to emit light through bioluminescence, were introduced into Salmonella typhimurium strain TA98. These bacteria were exposed for 48 h to chemicals or complex mixtures in 48-well microplates containing an appropriate liquid medium. Cells were subsequently centrifuged and resuspended in buffer. The final postexposure revertant biomass was then estimated using a microluminometer. Replication trials confirmed methodological reproducibility. Clear dose-response relationships were obtained with the direct frameshift mutagens 4NQO and 2NF. Mutagenicity threshold effect concentrations found for these compounds were comparable to those reported in the literature. Industrial effluents and environmental extracts (effluents, suspended solids) were also tested and results compared well with those of the SOS Chromotest. While further validation of this new adaptation of the Ames test will be required, it appears at this time that it could be well suited for routine screening of xenobiotics and environmental samples.

Bacterial bioluminescence: organization, regulation, and application of the lux genes.

Significant advances have been made in the characterization of luciferases and other lux-specific proteins as well as the lux genes from a number of different species of marine and terrestrial luminescent bacteria. A common lux gene organization (luxCDAB..E) modulated by the presence of specific genes involved in regulation and flavin binding and metabolism (luxF-I,L,R,Y) has been found with the luciferase genes (luxAB) flanked by the genes involved in synthesis of its fatty aldehyde substrate (luxCDE). For many species, light intensity per cell is highly dependent on cellular growth resulting in a spectacular autoinduction of luminescence at high cell density. Consequently, the bacterial lux system is of particular interest as it can serve as an excellent model for more general signal transduction systems involved in developmental processes, intercellular communication, and even symbioses. Identification of the lux autoinducers and regulatory proteins of Vibrio harveyi and Vibrio fischeri has provided the biochemical and genetic basis for dissection of the luminescent system. Isolation of the lux genes and the ability to transfer these genes into prokaryotic and eukaryotic organisms have greatly expanded the scope and potential uses of bacterial bioluminescence as a safe, rapid, and sensitive sensor for a wide variety of compounds and metabolic processes.

Molecular biology of bacterial bioluminescence.

The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.

Autoinduction of light emission in different species of bioluminescent bacteria.

The production of light in most luminescent bacteria lags behind cellular growth with induction of luminescence only occurring at high cell density. The maximum intensity and the cell density reached before induction of luminescence is highly dependent on the bacterial species and the growth conditions including temperature, salt and nutrient composition as well as other experimental conditions. Although common N-acylhomoserine lactone autoinducers have been identified in Vibrio (Photobacterium) fischeri and Vibrio harveyi, most regulatory components are quite different. Recognition of the common as well as the different elements controlling luminescence in the diverse bacterial species is essential to understand the basis for high levels of light emission at the later stages of cellular growth.

Purification and characterization of a poly(dA-dT) lux-specific DNA-binding protein from Vibrio harveyi and identification as LuxR.

A lux-specific DNA-binding protein was purified to homogeneity from Vibrio harveyi by chromatography on DEAE-Sepharose, DNA-cellulose, Superose 12, and Mono Q. A single polypeptide of M(r) = 23,000 was found on SDS-polyacrylamide gel electrophoresis with an amino-terminal sequence corresponding to that predicted for luxR, a gene that causes a shift in the transcriptional start site from position -123 to -26 base pairs upstream of the initiation codon of luxC in the V. harveyi lux operon and is required for high expression of lux mRNA in recombinant Escherichia coli. Identification of the DNA-binding protein as LuxR was confirmed by showing its absence in V. harveyi luxR-mutants and its synthesis in recombinant E. coli containing V. harveyi luxR. The LuxR protein was shown to bind to two specific (A + T)-rich regions of DNA upstream of the V. harveyi luxC gene: region A, -290 to -253 base pairs, and region B, -170 to -116 base pairs. Synthetic poly(dA-dT) but not poly(dA)-poly(dT) competed with the lux DNA for binding to LuxR suggesting that this protein may be a novel poly(dA-dT)-binding protein in prokaryotes. The LuxR protein inhibited transcription from the -123 promoter in vitro; however, transcription from the -26 promoter was not reconstituted suggesting the possible requirement for other factors in lux gene regulation. LuxR shared sequence identity with two proteins linked to the regulation of enzymes involved in electron transport indicating that it may be a member of a family of regulators of metabolic functions responsible for diverting electrons from the respiratory chain.

A lux-specific myristoyl transferase in luminescent bacteria related to eukaryotic serine esterases.

The diversion of fatty acids from fatty acid biosynthesis into the luminescent system is catalyzed by a lux-specific acyltransferase that catalyzes the cleavage of fatty acyl-acyl carrier protein (ACP). Analysis of the substrate specificities for fatty acyl-ACPs of the transferases from divergent luminescent bacteria, Photobacterium phosphoreum and Vibrio harveyi, has demonstrated that myristoyl-ACP is cleaved at the highest rate. Inhibition by phenylmethanesulfonyl fluoride as well as resistance of the acylated enzyme intermediate to cleavage by hydroxylamine showed that the transferase is a serine esterase. Moreover, activity was dependent on a basic residue with a pKa of 6.3 implicating a histidine residue as part of a charge relay system found in serine esterases. The nucleotide sequence of the P. phosphoreum luxD gene coding for the transferase was determined resulting in the identification of the active site motif for serine esterases, G-X-S-X-G. Replacement of the serine residue at the center of this motif by threonine, alanine, or glycine blocked the transferase acyl-ACP cleavage activity, its ability to be acylated, and complementation of a transferase defective mutant on transconjugation with the luxD gene. The sequence and location of the serine as well as a histidine residue in the lux-specific transferases were found to be similar to those involved in the charge relay system in vertebrate thioesterases. Combined with the similar kinetic properties, these results support a common metabolic role for both enzymes in the diversion of fatty acids from the fatty acid biosynthetic pathway.

The turnover of bacterial luciferase is limited by a slow decomposition of the ternary enzyme-product complex of luciferase, FMN, and fatty acid.

Bacterial luciferase catalyzes the conversion of reduced flavin mononucleotide, O2, and fatty aldehyde to FMN, H2O, and fatty acid with light being emitted at a very low rate characterized by the decay of luminescence in single turnover flash assays. The present studies have now revealed that the decomposition of the ternary complex of luciferase with FMN and myristic acid occurs at a rate 10-15 times slower than the decay of luminescence and that functional luciferase is only regenerated after the release of flavin. In contrast, the rate of FMN dissociation and recovery of activity observed with a binary luciferase-FMN complex was more rapid indicating that release of fatty acid played the critical role in determining the rate of FMN dissociation from the ternary complex. Decomposition of the ternary complex was shown to occur by an ordered process involving the slow release of fatty acid followed by the more rapid release of flavin. The present results suggest that the rate-limiting step for the turnover of luciferase occurs subsequent to emission of light and would be the controlling step under conditions (e.g. in cells) with continuous light emission.

An essential histidine residue required for fatty acylation and acyl transfer by myristoyltransferase from luminescent bacteria.

The lux-specific acyltransferases are serine esterases responsible for preferential diversion of myristic acid from fatty acid biosynthesis to the luminescent system. In contrast to other acyltransferases, an acylated enzyme intermediate can readily be detected making it ideal for the study of the mechanism of acyl transfer. Although the transferase readily cleaves acyl carrier protein and acyl-CoA, an alternate more rapid and convenient assay involving the cleavage of p-nitrophenyl acyl esters was developed and applied in these studies. The cleavage of the oxyesters by the transferase was shown to have a similar dependence on fatty acid chain length and organic solvents as the cleavage of thioesters. Using this assay, it could be demonstrated that the Photobacterium phosphoreum transferase was inactivated at pH 6 with diethyl pyrocarbonate at a rate (73 M-1 s-1, 10 degrees C) even faster than that reported for other enzymes with reactive histidyl residues at their active site. Spectral changes during chemical modification as well as restoration of activity by neutral hydroxylamine showed that the loss of activity was associated with modification of a single histidine residue. Replacement of the four histidine residues, conserved in all lux-specific acyltransferases, by asparagine demonstrated that cleavage of both thioesters and oxyesters by the P. phosphoreum acyltransferase as well as acylation of the enzyme was blocked on mutation of His-244 but not the other three conserved histidines (His-12, -52, and -75). These results suggest that the histidine residue near the carboxyl terminus (His-244) may be part of a catalytic triad essential for cleavage of acyl esters and transfer of the acyl group to the enzyme.

Complementation of subunits from different bacterial luciferases. Evidence for the role of the beta subunit in the bioluminescent mechanism.

Complementation of the nonidentical subunits (alpha and beta) of luciferases isolated from two different bioluminescent strains, Beneckea harveyi and Photobacterium phosphoreum, has resulted in the formation of a functional hybrid luciferase (alpha h beta p) containing the alpha subunit from B. harveyi luciferase (alpha h) and the beta subunit from P. phosphoreum luciferase (beta p). The complementation was unidirectional; activity could not be restored by complementing the alpha subunit of P. phosphoreum luciferase with the beta subunit of B. harveyi luciferase, showing that the subunits from these luciferases were not identical. Kinetic parameters of the hybrid luciferase reflecting the intermediate and later steps of the bioluminescent reaction as well as the overall activity and specificity were essentially identical to the same kinetic parameters for B. harveyi luciferase, the source of the alpha subunit, and quite distinct from those of P. phosphoreum luciferase. However, kinetic parameters that reflected the initial step in the reaction involving interaction of FMNH2 and luciferase were altered in the hybrid luciferase compared to both the parental luciferases, the Kd for FMNH2 actually being closer to that observed for the P. phosphoreum luciferase (the source of the beta subunit). These results provide direct evidence that modification or alteration of the beta subunit in a dimeric luciferase molecule can affect the kinetic properties and indicates that the beta subunit plays a functional role in the bioluminescent mechanism. It is proposed that both the alpha and beta subunits are involved with the initial interaction with FMNH2, whereas subsequent steps in the mechanism are dictated exclusively by the alpha subunit and are unaffected by alterations in the beta subunit.

Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence.

Pulse-chase experiments with [(3)H]tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from Vibrio harveyi can specifically catalyze the deacylation of a (3)H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this V. harveyi [(3)H]acylprotein and [(3)H]palmitoyl-ACP from Escherichia coli were substrates in vitro for either the V. harveyi 32-kDa acyltransferase or the analogous enzyme ("34K") from Photobacterium phosphoreum. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. Phosphate ions and, to a lesser extent, organic alcohols stimulated the rate of acyl-protein cleavage. No significant cleavage of either E. coli or V. harveyi tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence.