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At least 19 recordsLinked to original sources
PubMed2002

Mutational analysis of the subunit interface of Vibrio harveyi bacterial luciferase.

Bacterial luciferase is a heterodimeric (alphabeta) enzyme which catalyzes a light-producing reaction in Vibrio harveyi. In addition to the alphabeta enzyme, the beta subunit can self-associate to form a stable but inactive homodimer [Sinclair, J. F., Ziegler, M. M., and Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320-326]. The studies reported here were undertaken to explore the role of the subunit interface in the conformational stability of the enzyme. To this end, we constructed four mutant heterodimers in which residues at the subunit interface were changed in an effort to alter the volume of an apparent solvent accessible channel at the interface or to alter H-bonding groups. Equilibrium unfolding data for the heterodimer have been interpreted in terms of a three-state mechanism [Clark, C. A., Sinclair, J. F., and Baldwin, T. O. (1993) J. Biol. Chem. 268, 10773-10779]. However, we found that unfolding for the wild-type and mutant luciferases is better described by a four-state model. This change in the proposed mechanism of unfolding is based on observation of residual structure in the subunits following dissociation of the heterodimeric intermediate. All of the mutants display modest reductions in activity but, surprisingly, no change in the DeltaG2H2O value for subunit dissociation and no measurable change in the equilibrium dissociation constant relative to that of the wild-type heterodimer. However, the DeltaG1H2O value for the formation of the dimeric intermediate that precedes subunit dissociation is reduced for three of the mutants, indicating that mutations at the interface can alter the stability of a region of the alpha subunit that is distant from the interface. We conclude that the interface region communicates with the distal domains of this subunit, probably through the active center region of the enzyme.

Hydrogen Bonding↗
PubMed1999

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.

Aldehydes↗
PubMed1999

Folding, stability, and physical properties of the alpha subunit of bacterial luciferase.

Bacterial luciferase is a heterodimeric (alphabeta) enzyme composed of homologous subunits. When the Vibrio harveyi luxA gene is expressed in Escherichia coli, the alpha subunit accumulates to high levels. The alpha subunit has a well-defined near-UV circular dichroism spectrum and a higher intrinsic fluorescence than the heterodimer, demonstrating fluorescence quenching in the enzyme which is reduced in the free subunit [Sinclair, J. F., Waddle, J. J., Waddill, W. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036-5044]. Analytical ultracentrifugation of the alpha subunit has revealed a reversible monomer to dimer equilibrium with a dissociation constant of 14.9 +/- 4.0 microM at 18 degrees C in 50 mM phosphate and 100 mM NaCl, pH 7.0. The alpha subunit unfolded and refolded reversibly in urea-containing buffers by a three-state mechanism. The first transition occurred over the range of 0-2 M urea with an associated free-energy change of 2.24 +/- 0.25 kcal/mol at 18 degrees C in 50 mM phosphate buffer, pH 7.0. The second, occurring between 2.5 and 3.5 M urea, comprised a cooperative transition with a free-energy change of 6.50 +/- 0.75 kcal/mol. The intermediate species, populated maximally at ca. 2 M urea, has defined near-UV circular dichroism spectral properties distinct from either the native or the denatured states. The intrinsic fluorescence of the intermediate suggested that, although the quantum yield had decreased, the tryptophanyl residues remained largely buried. The far-UV circular dichroism spectrum of the intermediate indicated that it had lost ca. 40% of its native secondary structure. N-Terminal sequencing of the products of limited proteolysis of the intermediate showed that the C-terminal region of the alpha subunit became protease labile over the urea concentration range at which the intermediate was maximally populated. These observations have led us to propose an unfolding model in which the first transition is the unfolding of a C-terminal subdomain and the second transition represents the unfolding of a more stable N-terminal subdomain. Comparison of the structural properties of the unfolding intermediate using spectroscopic probes and limited proteolysis of the alpha subunit with those of the alphabeta heterodimer suggested that the unfolding pathway of the alpha subunit is the same, whether it is in the form of the free subunit or in the heterodimer.

Chemical Phenomena↗
PubMed1993

Kinetic destabilization of the hydroperoxy flavin intermediate by site-directed modification of the reactive thiol in bacterial luciferase.

Bacterial luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The reactive cysteinyl residue at position 106 of the alpha subunit has been replaced by serine, alanine, and valine by site-directed mutagenesis (Baldwin, T. O., Chen L. H., Chlumsky, L. J., Devine, J. H., and Ziegler, M. M. (1989) J. Biolumin. Chemilumin. 4, 40-48) and the kinetics of the reaction catalyzed by each mutant protein measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C. The time courses for the formation and decay of the various intermediates for the three alpha C106 mutants have been followed by monitoring the absorbance at 380 and 445 nm and the emission of visible light using n-decanal as the aldehyde substrate. The time courses for these events have been incorporated into a comprehensive kinetic model; 16 individual rate constants have been obtained for this model by numeric simulations of the time courses for the wild-type enzyme and for the three alpha C106 mutants. The mutants catalyzed the production of visible light demonstrating that the reactive thiol is not involved in the bioluminescence reaction. All three mutants have been found to catalyze the formation of the C4a-hydroperoxy flavin intermediate with rate constants equal to that of the wild-type enzyme. These results are incompatible with those reported by Xi et al. who have suggested that the major pathway for the oxidation of alpha C106V-bound FMNH2 does not involve the C4a-hydroperoxy flavin as an intermediate (Xi, L., Cho, K.-W., Herndon, M.E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203). The rates of decay of the C4a-hydroperoxy flavin intermediate with the mutant enzymes were found to be two orders of magnitude faster than that of the wild-type enzyme. Luciferase has been shown to be inhibited at high levels of aldehyde substrate when the enzyme is assayed by injecting FMNH2 into an aerobic mixture of enzyme and aldehyde. This aldehyde inhibition has been shown to occur by the formation of a dead-end enzyme-aldehyde complex which blocks the binding of FMNH2 to the enzyme; loss of activity is due to the rapid nonenzymatic decomposition of the reduced flavin with molecular oxygen.

Escherichia coli↗
PubMed1996

The 1.5-A resolution crystal structure of bacterial luciferase in low salt conditions.

Bacterial luciferase is a flavin monooxygenase that catalyzes the oxidation of a long-chain aldehyde and releases energy in the form of visible light. A new crystal form of luciferase cloned from Vibrio harveyi has been grown under low-salt concentrations, which diffract x-rays beyond 1.5-A resolution. The x-ray structure of bacterial luciferase has been refined to a conventional R-factor of 18.2% for all recorded synchrotron data between 30.0 and 1.50-A resolution. Bacterial luciferase is an alpha-beta heterodimer, and the individual subunits fold into a single domain (beta/alpha)8 barrel. The high resolution structure reveals a non-prolyl cis peptide bond that forms between Ala74 and Ala75 in the alpha subunit near the putative active site. This cis peptide bond may have functional significance for creating a cavity at the active site. Bacterial luciferase employs reduced flavin as a substrate rather than a cofactor. The structure presented was determined in the absence of substrates. A comparison of the structural similarities between luciferase and a nonfluorescent flavoprotein, which is expressed in the lux operon of one genus of bioluminescent bacteria, suggests that the two proteins originated from a common ancestor. However, the flavin binding sites of the nonfluorescent protein are likely not representative of the flavin binding site on luciferase. The structure presented here will furnish a detailed molecular model for all bacterial luciferases.

Amino Acid Sequence↗
PubMed2005

Implication of a critical residue (Glu175) in structure and function of bacterial luciferase.

Structural properties of a bacterial luciferase mutant, evolved by random mutagenesis, have been investigated. 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. By random mutagenesis, one of the mutants generated by a single mutation on LuxA at position 175 (E175G) resulted in the "slow decay" Xenorhabdus luminescens luciferase was converted into a luciferase with a significantly more rapid decay rate [Hosseinkhani, S., Szittner, R. and Meighen, E.A. (2005) Biochemical Journal 385, 575-580]. A single mutation (E175G), in a loop that connects alpha helix 5 and beta sheet 5 brought about changes in the kinetic and structural properties of the enzyme. Enhancement of tryptophan fluorescence was observed with a lower degree of fluorescence quenching by acrylamide upon mutation. Near- and far-UV circular dichroism spectra of the native and mutant forms suggested formation of an intermediate structure, further supported by 8-anilino-1-naphthalene-sulphonic acid (ANS) fluorescence which indicated lower exposure of hydrophobic residues as a result of mutation. Fluorescence quenching studies utilizing acrylamide indicated a more accessible fluor for the native form. Thus, the E175G point mutation appears to change the enzymatic decay rate by inducing a substantial tertiary structural change, without a large effect on secondary structural elements, as revealed by Fourier transform IR spectroscopy. Overall, the mutation caused structural changes that go beyond the simple change in orientation of Glu175.

Animals↗
PubMed2001

Functional implications of the unstructured loop in the (beta/alpha)(8) barrel structure of the bacterial luciferase alpha subunit.

Bacterial luciferase catalyzes the conversion of FMNH(2), a long-chain aliphatic aldehyde, and molecular oxygen to FMN, the corresponding carboxylic acid, and H(2)O with the emission of light. The light-emitting species is an enzyme-bound excited state flavin. The enzyme is a heterodimer (alphabeta) of homologous subunits each with an (beta/alpha)(8) barrel structure. A portion of the loop in the alpha subunit that connects beta strand 7 to alpha helix 7 is disordered in the crystal structure. To test the hypothesis that this loop closes over the active site during catalysis and protects the active site from bulk solvent, a mutant was constructed in which the 29 residues that are disordered in the 2.4 A crystal structure were deleted. Deletion of this loop results in a heterodimer with a subunit equilibrium dissociation constant of 1.32 +/- 1.25 microM, whereas the wild-type heterodimer shows no measurable subunit dissociation. This mutant retains its ability to bind substrate flavin and aldehyde with wild-type affinity and can carry out the chemistry of the bioluminescence reaction with nearly wild-type efficiency. However, the bioluminescent quantum yield of the reaction is reduced nearly 2 orders of magnitude from that of the wild-type enzyme.

Binding Sites↗
PubMed1988

The use of the luxA gene of the bacterial luciferase operon as a reporter gene.

Bacterial luciferase can be assayed rapidly and with high sensitivity both in vivo and in vitro. Here we demonstrate that the N-terminal hydrophobic domain of the alpha catalytic subunit of the luciferase enzyme is indispensable for enzyme activity, although N-terminal translational fusions with full luciferase activity can be obtained. Bacterial luciferase is therefore ideally suited as a reporter enzyme for gene fusion experiments. A list of vectors for the convenient use of the luciferase marker genes to monitor gene expression in vivo are presented.

Base Sequence↗
PubMed1993

GroE-mediated folding of bacterial luciferases in vivo.

In this study we present evidence indicating that GroE chaperonins mediate de novo protein folding of heterodimeric and monomeric luciferases under heat shock or sub-heat shock conditions in vivo. The effects of additional groESL and groEL genes on the bioluminescence of Escherichia coli cells expressing different bacterial luciferase genes at various temperatures were directly studied in cells growing in liquid culture. Data indicate that at 42 degrees C GroESL chaperonins are required for the folding of the beta subunit polypeptide of the heterodimeric alpha beta luciferase from the mesophilic bacterium Vibrio harveyi MAV (B392). In contrast, the small number of amino acid substitutions present in the luciferase beta subunit polypeptide from the thermotolerant V. harveyi CTP5 suppresses this requirement for GroE chaperonins, and greatly reduces interaction between the beta subunit polypeptide and GroEL chaperonin. In addition, GroESL are required for the de novo folding at 37 degrees C of a MAV alpha beta luciferase fusion polypeptide that is functional as a monomer. No such requirement for luciferase activity is observed at that temperature with a fusion of the CTP5 alpha and beta subunit polypeptides, although GroE chaperonins can still mediate folding of the CTP5 fusion luciferase. Bacterial luciferases provide a unique system for direct observation of the effects of GroE chaperonins on protein folding and enzyme assembly in living cells. Furthermore, they offer a sensitive and simple assay system for the identification of polypeptide domains required for GroEL protein binding.

Amino Acid Sequence↗
PubMed1993

Interaction of bacterial luciferase with aldehyde substrates and inhibitors.

Bacterial luciferase catalyzes the reaction of FMNH2, O2, and an aliphatic aldehyde to yield the carboxylic acid, FMN, water and blue-green light. The kinetics of the bacterial luciferase reaction were measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C for the series of aldehydes from n-heptanal to n-undecanal. The rate of formation of the 4a-hydroperoxyflavin intermediate was dependent on the aldehyde concentration when mixtures of enzyme, FMNH2, and aldehyde were rapidly mixed with O2. At saturating aldehyde, the rate of formation of this intermediate was 100-fold slower than in the absence of aldehyde, demonstrating that an enzyme.FMNH2.aldehyde complex can be formed. Numerical simulation of the time courses for these experiments supported the formation of this intermediate and its direct reaction with O2. The kinetics of the light emitting reaction were dependent upon the chain length of the aldehyde substrate. Although the initial light intensity and the light emission decay rate were different for each aldehyde, the quantum yield for the reaction was independent of the aldehyde used. Luciferase was inhibited by high levels of the aldehyde substrate when the enzyme was assayed by mixing FMNH2 with an aerobic mixture of enzyme and aldehyde. The extent of inhibition was dependent on the particular aldehyde used, and the binding affinity of the aldehyde for the free enzyme increased in parallel with the aldehyde chain length. The kinetics of the formation and decay of the various intermediates were also studied in the presence of n-alkyladehyde analogs. These compounds decreased the rate of formation of the 4a-hydroperoxyflavin intermediate in much the same way as the aldehyde substrate, presumably by the formation of the enzyme.FMNH2.analog ternary complex.

Aldehydes↗
PubMed1975

Differential effects of 8-anilino-1-naphthalenesulfonate upon binding of oxidized and reduced flavines by bacterial luciferase.

Upon binding to bacterial luciferase, both the absorption and the fluorescence excitatiom maxima of 8-anilino-1-naphthalensulfonate (ANS) shift from 353 to 370 nm while the fluorescence emission optimum shifts from 540 to 480 nm, and the fluorescence quantum yield increases from 0.003 to 0.39, indicating that the environment of the ANS binding site is hydrophobic. ANS binds to luciferase with dissociation constants of 1.9 X 10(-5) and 2.3 X 10(-5) M at 5 and 23 degrees, repsectively. As with both oxidized flavine mononucleotide (FMN) and reduced flavine mononucleotide (FMNH2), ANS also binds to luciferase with a stoichiometry of 1 site per dimeric luciferase molecule. ANS acts as a luciferase inhibitor, competitive with FMNH2, with an inhibitor constant of 2.3 X 10(-5) M at 23 degrees. However, the binding of ANS does not significantly displace FMN from binding to luciferase. Interactions of FMN and FMNH2 with luciferase are thus differentially regulated by the ANS binding.

Anilino Naphthalenesulfonates↗
PubMed1980

The determination of reduced nicotinamide-adenine dinucleotide and metabolic intermediates in picomole amounts with bacterial luciferase.

Methods that use bacterial luciferase for the assay of NADH in the range from 1 pmol to 1 nmol are described. Optimal conditions for the assay of glycolytic intermediates, tricarboxylic acid-cycle intermediates and related amino acids from milligram amounts of tissue are presented. The whole spectrum of these intermediates can be determined on about 10 mg of liver tissue. The methods are simple, are suitable for routine use, and the instrumentation is inexpensive. The concentrations of glycolytic intermediates in rat livers were determined by conventional spectrometric methods and with luciferase, and the results found to be in good agreement.

Animals↗
PubMed1994

Thermostability of bacterial luciferase expressed in different microbes.

Bacterial luciferase was used to investigate the relationship between the thermostability of a cytoplasmic reporter molecule and cellular heat resistance. The luciferase activity of Vibrio fischeri was expressed in strains of Escherichia coli, Salmonella typhimurium, Listeria monocytogenes and Brochothrix thermosphacta following transformation with plasmid pSP13 carrying the luxAB genes. The thermostability of intracellular luciferase varied depending on the organism in which it was expressed, but was not related to the cellular heat resistance of the different organisms. Addition of xylitol to the heating medium protected against loss of viability and inactivation of intracellular luciferase. Glycerol also protected against loss of viability but was less effective at preventing thermal denaturation of luciferase.

Culture Media↗
PubMedDate not supplied

[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.

Adenosine Triphosphatases↗
PubMed1994

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.

Enzyme Stability↗
PubMed1992

Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae.

Bacterial luciferase, derived from a fusion of the luxA and luxB genes of Vibrio harveyi, has been expressed at very high levels in caterpillars and insect cells. The coding sequence for luciferase was inserted into vectors developed in our laboratory which were designed to expedite screening of recombinant virus. These vectors contained the beta-galactosidase indicator gene under control of immediate early (IE1), early (ETL), or very late (P10) promoters and a cloning site for inserting the fused luciferase gene next to the polyhedrin promoter. Recombinant baculoviruses containing the luciferase gene as well as the beta-galactosidase gene could be easily selected when Bluo-gal (beta-galactosidase indicator) was included in the plaque assays. Using cells derived from the fall armyworm (Spodoptera frugiperda), luciferase was strongly expressed very late in infection (48-72 h). The bacterial luciferase assay was sufficiently sensitive that light production could be detected from an extract of a single cell. In addition, live insects, including the cabbage looper (Trichoplusia ni) and saltmarsh caterpillar (Estigmene acrea) were infected by mixing recombinant baculovirus into their diet. Cabbage loopers (with an average wet weight of 223 mg) produced at least 195 micrograms of active luciferase and levels of synthesis peaked between 96-120 h. The results indicate that bacterial luciferase may be used as a reporter of gene expression in insects.

Animals↗