Random and site-directed mutagenesis of bacterial luciferase: investigation of the aldehyde binding site

Numerous luciferase structural gene mutants of Vibrio harveyi have been generated by random mutagenesis and phenotypically characterized [Cline, T.W., & Hastings, J.W. (1972) Biochemistry 11, 3359-3370]. All mutants selected by Cline and Hastings for altered kinetics in the bioluminescence reaction had lesions in the alpha subunit. One of these mutants, AK-20, has normal or slightly enhanced thermal stability and enhanced FMNH2 binding affinity but a much-reduced quantum yield of bioluminescence and dramatically altered stability of the aldehyde-C4a-peroxydihydroflavin-luciferase intermediate (IIA), with a different aldehyde chain length dependence from that of the wild-type luciferase. To better understand the structural aspects of the aldehyde binding site in bacterial luciferase, we have cloned the luxAB genes from the V. harveyi mutant AK-20, determined the nucleotide sequence of the entire luxA gene, and determined the mutation to be TCT----TTT, resulting in a change of serine----phenylalanine at position 227 of the alpha subunit. To confirm that this alteration caused the altered kinetic properties of AK-20, we reverted the AK-20 luxA gene by oligonucleotide-directed site-specific mutagenesis to the wild-type sequence and found that the resulting enzyme is indistinguishable from the wild-type luciferase with respect to quantum yield, FMNH2 binding affinity, and intermediate IIA decay rates with 1-octanal, 1-decanal, and 1-dodecanal. To investigate the cause of the AK-20 phenotype, i.e., whether the phenotype is due to loss of the seryl residue or to the properties of the phenylalanyl residue, we have constructed mutants with alanine, tyrosine, and tryptophan at alpha 227.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity coupling and complex formation between bacterial luciferase and flavin reductases.

Luminous bacteria contain several species of flavin reductases, which catalyze the reduction of FMN using NADH and/or NADPH as a reductant. The reduced FMN (i.e. FMNH(2)) so generated is utilized along with a long-chain aliphatic aldehyde and molecular oxygen by luciferase as substrates for the bioluminescence reaction. In this report, the general properties of luciferases and reductases from luminous bacteria are briefly summarized. Earlier and more recent studies demonstrating the direct transfer of FMNH(2) from reductases to luciferase are surveyed. Using reductases and luciferases from Vibrio harveyi and Vibrio fischeri, two mechanisms were uncovered for the direct transfer of reduced flavin cofactor and reduced flavin product of reductase to luciferase. A complex of an NADPH-specific reductase (FRP(Vh)) and luciferase from V. harveyi has been detected in vitro and in vivo. Both constituent enzymes in such a complex are catalytically active. The reduction of FRP(Vh)-bound FMN cofactor by NADPH is reversible, allowing the cellular contents of NADP(+) and NADPH as a factor for the regulation of the production of FMNH(2) by FRP(Vh) for luciferase bioluminescence. Other regulations of the activity coupling between reductase and luciferase are also discussed.     

Recovery of components of fluorescence spectra of mixtures by intensity- and anisotropy decay-associated analysis: the bacterial luciferase intermediates

Reaction of FMNH2 and O2 with bacterial luciferase followed by blue light irradiation results in a product previously claimed to have the same fluorescence spectral distribution as the bioluminescence. Preparations of this "high fluorescence" intermediate, however, contain two fluorescent components, one from the intermediate and the other its breakdown product, FMN. Since the intermediate has a fluorescence lifetime of around 10 ns and a rotational correlation time in the range of 100 ns, compared to 5.0 and 0.15 ns, respectively, for the FMN, the two components can be successfully resolved from the total fluorescence by an anisotropy decay- and fluorescence decay-associated analysis employing simultaneous global computational methods. The fluorescence spectra of the intermediates from two types of luciferase were analyzed in this way; one luciferase was from Vibrio harveyi and the other was from an unusual type of V. fischeri that had an in vivo bioluminescence maximum at 505 nm, a wavelength almost 20 nm longer than that of the V. harveyi bioluminescence. For V. harveyi the true fluorescence of the intermediate is distinct from the bioluminescence, being found at a wavelength about 10 nm longer. For the type of V. fischeri examined, any difference in the two spectra is less certain. A control experiment with the dye 8-amino-1- naphthalenesulfonate bound to BSA and mixed with FMN recovered the original spectrum of the bound dye accurately.     

Relationship between the conserved alpha subunit arginine 107 and effects of phosphate on the activity and stability of Vibrio harveyi luciferase.

The Arg107 of the alpha subunit is a conserved residue for all known bacterial luciferases. The phosphate moiety of the reduced flavin mononucleotide (FMNH(2)) side chain has been hypothesized to be anchored at this site (A. J. Fisher, F. M. Raushel, T. O. Baldwin, and I. Rayment Biochemistry 34, 6581-6586, 1995). Mutations of alphaArg107 of the Vibrio harveyi luciferase to alanine, serine, and glutamate were carried out to test such a hypothesis. These variants were characterized and compared with the wild-type luciferase with respect to their K(m) for decanal, FMNH(2), and reduced riboflavin in both low- (0.01 or 0.05 M) and high- (0.3 M) phosphate buffers at pH 7.0. Results are consistent with the hypothesized binding of the FMNH(2) phosphate group by alphaArg107. Moreover, the alphaArg107 residue was apparently important in the expression of the luciferase maximal activity and aldehyde binding. Phosphate ion is also known to have other effects on luciferase stability. We compared the three luciferase variants with the native enzyme with respect to the decay rate of the FMN 4a-hydroperoxide intermediate II, and rates of inactivation by trypsin digestion, modification by N-ethylmaleimide, and heat treatment in low- and high-phosphate buffers. On the basis of patterns of the phosphate effects, alphaArg107 appeared to be important to the enhancement of luciferase stability against trypsin proteolysis at high phosphate but was not involved in regulating the intermediate II decay or sensitivity to N-ethylmaleimide modification. Differential effects of mutations on luciferase thermal stability were observed. It is uncertain whether alphaArg107 is involved in the enhanced thermal stability of the native luciferase in high phosphate buffer.     

The effect of Clp proteins on DnaK-dependent refolding of bacterial luciferases

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK-DnaJ-GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock.     

Cloning and expression of the Photobacterium phosphoreum luminescence system demonstrates a unique lux gene organization

The organization of the lux structural genes (A-E) in Photobacterium phosphoreum has been determined and a new gene designated as luxF discovered. The P. phosphoreum luminescence system was cloned into Escherichia coli using a pBR322 vector and identified by cross-hybridization with Vibrio fischeri lux DNA. The lux genes were located by specific expression of P. phosphoreum DNA fragments in the T7-phage polymerase/promoter system in E. coli and identification of the labeled polypeptide products. The luxA and luxB gene products (luciferase subunits) were shown to catalyze light emission in the presence of FMNH2, O2, and aldehyde. The luxC, luxD, and luxE gene products (fatty acid reductase subunits) responsible for aldehyde biosynthesis could be specifically acylated with 3H-labeled fatty acids. The order of the lux genes in P. phosphoreum was found to be luxCDABFE with luxF coding for a new polypeptide of 26 kDa. The presence of a new gene in the P. phosphoreum luminescence system between luxB and luxE as compared to the organization of the lux structural gene in V. fischeri and Vibrio harveyi (luxCDABE) demonstrates that the luminescent systems in the marine bacteria have significantly diverged. The discovery of the luxF gene provides the basis for elucidating the role of its gene product in the expression of luminescence in different marine bacteria.     

Localisation of luciferase in luminous marine bacteria by gold immunocytochemical labelling

Abstract Bioluminescent bacteria are able to produce light by an oxidative reaction involving the enzyme luciferase. The biochemistry of the reaction has been well documented. It has been postulated from the reaction pathway that the enzyme luciferase is membrane bound. Two species of luminous bacteria, Vibrio fischeri and V. harveyi , were isolated from sea water, fixed and embedded in resin for transmission electron microscopy. A commercially available V. fischeri bacterial luciferase was used to immunise rats for production of an antibody against luciferase. This antibody was coupled with a protein A gold probe and used to immunolabel the sections of bacteria. It was shown that the luciferase was associated with the inner membrane, and to a lesser extent present in the cytoplasm. Our results confirm that the luciferase is predominantly membrane bound.     

Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis.

Genes encoding NAD(P)H-flavin oxidoreductases (flavin reductases) similar in both size and sequence to Fre, the most abundant flavin reductase in Escherichia coli, were identified in four species of luminous bacteria, Photorhabdus luminescens (ATCC 29999), Vibrio fischeri (ATCC 7744), Vibrio harveyi (ATCC 33843), and Vibrio orientalis (ATCC 33934). Nucleotide sequence analysis showed Fre-like flavin reductases in P. luminescens and V. fischeri to consist of 233 and 236 amino acids, respectively. As in E. coli Fre, Fre-like enzymes in luminous bacteria preferably used riboflavin as an electron acceptor when NADPH was used as an electron donor. These enzymes also were good suppliers of reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. In V. fischeri, the Fre-like enzyme is a minor flavin reductase representing < 10% of the total FMN reductase. That the V. fischeri Fre-like enzyme has no appreciable homology in amino acid sequence to the major flavin reductase in V. fischeri, FRase I, indicates that at least two different types of flavin reductases supply FMNH2 to the luminescence system in V. fischeri. Although Fre-like flavin reductases are highly similar in sequence to luxG gene products (LuxGs), Fre-like flavin reductases and LuxGs appear to constitute two separate groups of flavin-associated proteins.     

Electronic excitation transfer in the complex of lumazine protein with bacterial bioluminescence intermediates

Fluorescence dynamics measurements have been made on the bioluminescence reaction intermediates using Photobacterium leiognathi, Vibrio fischeri, and Vibrio harveyi luciferases, both alone and in mixtures with Photobacterium phosphoreum lumazine protein. Each luciferase produces a "fluorescent transient" intermediate on reaction with the bioluminescence substrates, FMNH2, tetradecanal, and O2, and all have a fluorescence quantum yield about 0.3, with a predominant lifetime around 10 ns. The P. leiognathi luciferase fluorescent transient has a rotational correlation time of 79 ns at 2 degrees C, as expected for the rotational diffusion of a 77-kDa macromolecule. In the presence of lumazine protein however a faster correlation time of about 3 ns predominates. This rapid channel of anisotropy loss is attributed to energy transfer from the flavin intermediate bound on the luciferase to the lumazine ligand, reflects the presence of protein-protein complexation, and is greatest in the case of P. leiognathi, but not at all for V. fischeri. This fact is consistent with the strong influence of lumazine protein on the bioluminescence reaction of P. leiognathi, and not at all with V. fischeri. The rate of energy transfer is of order 10(9) s-1, much greater than the 10(8) s-1 fluorescence rate of the donor. Thus the bioluminescence excitation of lumazine protein could occur by a similar photophysical mechanism of interprotein energy transfer from a chemically excited fluorescent transient donor to the lumazine acceptor.     

Expression of bioluminescence by Escherichia coli containing recombinant Vibrio harveyi DNA.

When isogenic strains of Escherichia coli, RR1 (rec+) and HB101 (recA), were transformed with mapped recombinant plasmids known to contain Vibrio harveyi luciferase genes and large regions of DNA flanking on both sides, a small percentage (0.005%) of the colonies expressed high levels of luminescence (up to 10(12) quanta s-1 ml-1) in the absence of added aldehyde. The altered ability to express light was found to be due to a mutation in the host and not to an alteration in the recombinant DNA. When these bright colonies were cured of plasmid, they could be retransformed with cloned V. harveyi gene fragments in cis and in trans to yield luminescent colonies at 100% frequency. The maximum length of V. harveyi DNA required to produce light-emitting E. coli was shorter (6.3 kilobase pairs) than that required for expression of the V. fischeri system in E. coli. Cell extracts from bright clones contained wild-type levels of activity for the heteropolymeric (alpha beta) luciferase; fatty acid labeling revealed the presence of the three acylated polypeptides of the fatty acid reductase system which is involved in aldehyde biosynthesis for the luminescence reaction. The increased light emission in the mutant bacteria appeared to arise in part from production of higher levels of polycistronic mRNAs coding for luciferase.     

The nucleotide sequence of the luxE gene of Vibrio harveyi and a comparison of the amino acid sequences of the acyl-protein synthetases from V. harveyi and V. fischeri

The luxE gene of bioluminescent bacteria encodes the acyl-protein synthetase component of the fatty acid reductase complex. The complex is responsible for converting tetradecanoic acid to the aldehyde which serves as a substrate in the luciferase-catalyzed reaction. The nucleotide sequence of the luxE gene of Vibrio harveyi was determined and the amino acid sequence of the acyl-protein synthetase deduced. The protein consists of 378 amino acid residues and has a molecular weight of 42,965 daltons. Alignment of the V. harveyi enzyme with the V. fischeri acyl-protein synthetase showed 62% identity.     

A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.     

Bioluminescence spectral and fluorescence dynamics study of the interaction of lumazine protein with the intermediates of bacterial luciferase bioluminescence

The mechanism of the shifting of the bioluminescence spectrum from the reaction of bacterial luciferase by lumazine protein is investigated by methods of fluorescence dynamics. A metastable intermediate is produced on reaction of Vibrio harveyi luciferase with FMNH2 and O2. It has an absorption maximum at 374 nm and a rotational correlation time (phi) derived from the decay of its fluorescence (maximum 500 nm) anisotropy of 90 ns (2 degrees C). Lumazine protein from Photobacterium phosphoreum has an absorption maximum at 417 nm and a fluorescence maximum at 475 nm. Lumazine protein forms a protein-protein complex with luciferase, and the complex has a phi of approximately 100 ns. A mixture of lumazine protein and the intermediate would be expected to have an average correlation time (phi av) around 100 ns, but instead, the result is anomalous. The phi av is much lower and is also wavelength dependent. For excitation at 375 nm, which is mainly absorbed in the flavin chromophore of the intermediate, phi av = 25 ns, but at 415 nm, mainly absorbed by the lumazine derivative ligand of lumazine protein, phi av approximately 50 ns. It is proposed that protein-protein complexation occurs between lumazine protein and the luciferase intermediate and that in this complex energy transfer from the flavin to the lumazine is the predominant channel of anisotropy loss. A distance of 20 A between the donor and acceptor is calculated. In the bioluminescence reaction of intermediate with tetradecanal, a fluorescent transient species is produced which is the bioluminescence emitter.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Luciferase from Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues

A new luciferase from V. campbellii (Lux_Vc) was cloned and expressed in Escherichia coli and purified to homogeneity. Although the amino acid sequences and the catalytic reactions of Lux_Vc are highly similar to those of the luciferase from V. harveyi (Lux_Vh), the two enzymes have different affinities toward reduced FMN (FMNH(-)). The catalytic reactions of Lux_Vc and Lux Vh were monitored by stopped-flow absorbance and luminescence spectroscopy at 4 degrees C and pH 8. The measured Kd at 4 degrees C for the binding of FMNH(-) to Lux_Vc was 1.8 microM whereas to Lux_Vh, it was 11 microM. Another difference between the two enzymes is that Lux_Vc is more stable than Lux_Vh over a range of temperatures; Lux_Vc has t1/2 of 1020 min while Lux_Vh has t1/2 of 201 min at 37 degrees C. The superior thermostability and tighter binding of FMNH(-) make Lux_Vc a more tractable luciferase than Lux_Vh for further structural and functional studies, as well as a more suitable enzyme for some applications. The kinetics results reported here reveal transient states in the reaction of luciferase that have not been documented before.     

A cyanide-aldehyde complex inhibits bacterial luciferase.

Cyanide at high (millimolar) concentrations inhibited in the in vitro Vibrio harveyi luciferase reaction. Cyanide reacted with free aldehyde to form an inhibitor. Inhibitor formation was accelerated by alkaline conditions and bovine serum albumin.     

Cloning and nucleotide sequence of luxR, a regulatory gene controlling bioluminescence in Vibrio harveyi.

Mutagenesis with transposon mini-Mulac was used previously to identify a regulatory locus necessary for expression of bioluminescence genes, lux, in Vibrio harveyi (M. Martin, R. Showalter, and M. Silverman, J. Bacteriol. 171:2406-2414, 1989). Mutants with transposon insertions in this regulatory locus were used to construct a hybridization probe which was used in this study to detect recombinants in a cosmid library containing the homologous DNA. Recombinant cosmids with this DNA stimulated expression of the genes encoding enzymes for luminescence, i.e., the luxCDABE operon, which were positioned in trans on a compatible replicon in Escherichia coli. Transposon mutagenesis and analysis of the DNA sequence of the cloned DNA indicated that regulatory function resided in a single gene of about 0.6-kilobases named luxR. Expression of bioluminescence in V. harveyi and in the fish light-organ symbiont Vibrio fischeri is controlled by density-sensing mechanisms involving the accumulation of small signal molecules called autoinducers, but similarity of the two luminescence systems at the molecular level was not apparent in this study. The amino acid sequence of the LuxR product of V. harveyi, which indicates a structural relationship to some DNA-binding proteins, is not similar to the sequence of the protein that regulates expression of luminescence in V. fischeri. In addition, reconstitution of autoinducer-controlled luminescence in recombinant E. coli, already achieved with lux genes cloned from V. fischeri, was not accomplished with the isolation of luxR from V. harveyi, suggesting a requirement for an additional regulatory component.     

Differential regulation of enzyme activities involved in aldehyde metabolism in the luminescent bacterium Vibrio harveyi.

The effects of catabolite repression and nutrient abundance on the activities of Vibrio harveyi enzymes known to be related to aldehyde metabolism were investigated. The growth of cells in complex medium containing glucose, which decreases in vivo luminescence and luciferase synthesis, also resulted in decreases in the specific activities of V. harveyi aldehyde dehydrogenase and acyl carrier protein acyltransferase as well as in the degree of fatty acylation of three bioluminescence-specific polypeptides (32, 42, and 57 kilodaltons), as monitored by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This repression was partially alleviated in glucose medium containing cyclic AMP. The acylation of the above-mentioned proteins, in addition to light emission and luciferase and acyltransferase activities, was also repressed when cells were grown in minimal medium, with partial recovery of these functions upon the addition of arginine. In contrast, aldehyde dehydrogenase activity was increased in minimal medium. These results suggest that the 42-, 57-, and 32-kilodalton proteins, which are responsible for the supply and reduction of fatty acids to form aldehydes for the luciferase reaction, are regulated in the same way as luciferase under the above-described conditions. However, aldehyde dehydrogenase, whose role in V. harveyi aldehyde metabolism is not yet known, is regulated in a different way with respect to nutrient composition.