Structurally distinct bacterial luciferases

Hastings, J. Woodland; Weber, K; Friedland, Joan; Eberhard, Anatol; Mitchell, George W.; Gunsalus, Androbert P.

doi:10.1021/bi00840a004

Cited by 152 publications

(107 citation statements)

References 17 publications

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“…Accordingly, dodecanal and octanal were selected as aldehydes (Hastings et al, 1969), and experiments were performed at a lower temperature (-4 "C).…”

Section: Introductionmentioning

confidence: 99%

Spectral detection of an intermediate preceding the excited state in the bacterial luciferase reaction

1993

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ABSTRACT:The bioluminescent reaction of luciferase isolated from Vibrio harveyi, strain M17, was initiated by mixing the luciferase-bound flavin 4a-hydroperoxide intermediate, purified in advance, with a long-chain aldehyde (dodecanal or octanal) at -4 O C . Measurements of absorbance changes from 300 to 600 nm during the course of the reaction revealed the existence of three sets of isosbestic points and three kinetic phases, the second of which parallels kinetically the decay of bioluminescence, measured concurrently. The absorbance changes in this second step and the decay of light emission exhibited similar deuterium isotope effects; this is postulated to be the step giving rise to the excited state and the enzyme-bound flavin 4a-hydroxide. The first step of the reaction, however, did not show an isotope effect; the intermediate thereby formed, observed here for the first time, is postulated to correspond to the luciferase-bound flavin 4a-peroxyhemiacetal.Of the many light-emitting organisms, only the bacteria produce a luciferase that utilizes a flavin as a substrate and the emitter (Hastings, 1983). Bacterial luciferase (EC 1.14.14.3) is a monooxygenase which gives rise to light emission upon reaction of enzyme-bound FMNH2' with molecular oxygen and a long-chain aldehyde. The products are oxidized FMN, the corresponding fatty acid, and water (Hastings et al., 1985;Baldwin & Ziegler, 1991). Thepostulatedpathway and principal intermediates (all luciferase-bound) are shown in Scheme 1.A key intermediate (11), which is formed by reaction of FMNH2 with oxygen and occurs prior to reaction with aldehyde, is the flavin 4a-hydroperoxide (Hastings et al., 1973;Vervoort et al., 1986). An intermediate formed concurrently with light emission (IV) was isolated and characterized by Kurfiirst et al. (1984Kurfiirst et al. ( ,1987. However, an intermediate (111) postulated to occur upon reaction of I1 with aldehyde via the Michaelis complex (IIA), but preceding light emission, remained to be demonstrated.Since Kurfiirst et al. (1984) used decanal, a fast-reacting aldehyde, we thought it possible that the existence of this earlier intermediate might be easier to detect under conditions where the reaction proceeds more slowly. Accordingly, dodecanal and octanal were selected as aldehydes (Hastings et al., 1969), and experiments were performed at a lower temperature (-4 "C).The reactions were carried out starting with the luciferasebound flavin 4a-hydroperoxide (11) prepared and purified in advance. Concurrent measurements of absorption and light emission were made. The results show three sets of isosbestic points, indicating the existence of an additional intermediate, which is postulated to correspond to the luciferase flavin 4a- Abbreviations: FMN and FMNH2, riboflavin 5'-phosphate and its reduced form; NMR, nuclear magnetic resonance.

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“…Accordingly, dodecanal and octanal were selected as aldehydes (Hastings et al, 1969), and experiments were performed at a lower temperature (-4 "C).…”

Section: Introductionmentioning

confidence: 99%

Spectral detection of an intermediate preceding the excited state in the bacterial luciferase reaction

1993

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“…Luciferases from the marine bacteria Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi, and Photobacterium phosphoreum are all a1 heterodimers (14,19,25), with molecular weights of approximately 40,000 for a and 37,000 for P3. For the former three species, the luxA and luxB genes, encoding the luciferase a and P subunits, respectively, have been sequenced (4,9,15,17).…”

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Cloning and nucleotide sequences of lux genes and characterization of luciferase of Xenorhabdus luminescens from a human wound

Cho

1991

J Bacteriol

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Xenorhabdus luminescens HW is the only known luminous bacterium isolated from a human (wound) source. A recombinant plasmid was constructed that contained the X. luminescens HW luxA and luxB genes, encoding the luciferase a and j subunits, respectively, as well as luxC, luxD, and a portion of luxE. The nucleotide sequences of these lux genes, organized in the order luxCDABE, were determined, and overexpression of the cloned luciferase genes was achieved in Escherichia coli host cells. The cloned luciferase was indistinguishable from the wild-type enzyme in its in vitro bioluminescence kinetic properties. Contrary to an earlier report, our findings indicate'that neither the specific activity nor the size of the a (362 amino acid residues, Mr 41,389) and 1B(324 amino acid residues, M, 37,112) subunits of the X. luminescens HW luciferase was unusual among known luminous bacterial systems. Significant sequence homologies of the a and ,B subunits of the X. luminescens HW luciferase with those of other luminous bacteria were observed. However, the X. luminescens HW luciferase was unusual in the high stability of the 4a-hydroperoxyflavin intermediate and its sensitivity to aldehyde substrate inhibition.Most of the known luminous bacteria are of marine origin and fall within two genera, Vibrio and Photobacterium (references 13 and 23'and references therein). Luciferases from the marine bacteria Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi, and Photobacterium phosphoreum are all a1 heterodimers (14,19,25), with molecular weights of approximately 40,000 for a and 37,000 for P3. For the former three species, the luxA and luxB genes, encoding the luciferase a and P subunits, respectively, have been sequenced (4,9,15,17).A few nonmarine luminous bacterial species have also been identified (13, 23); among them are Xenorhabdus luminescens Hb and Hm, both of which are insect-pathogenic symbionts with nematodes of the family Heterorhabditidae (24,27,30). The luciferases from X. luminescens Hm and Hb have identical a subunits and only a single different amino acid residue in the P subunit. Moreover, they are similar in specific activity and subunit sizes to and have significant sequence homology with luciferases from marine luminous bacteria (16, 27, 29). The luxA and luxB genes from these two X. luminescens strains differ by only one and two bases, respectively (16,29). In fact, the known sequences of the lux operon, starting with the last 142 bases of luxD through the first four bases of luxE (with luxA, luxB, and noncoding regions in between) show 99% identity for X. luminescens Hm and Hb (16,29).A different luminous strain of X. luminescens has been isolated from a human wound (8), now designated strain HW. Some unusual properties have been reported for this strain and its luciferase (5) in comparison with all other known luminous bacteria, including X. luminescens Hm and Hb. X. luminescens HW bioluminescence activities both in vivo and in vitro have been found to be substantially lower in intensity but higher i...

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“…However, based on the report that the quantum yield with respect to FMNH2 is approximately half that for aldehyde, it again was proposed that two reduced flavins are required per catalytic cycle (16,17 MATERIALS AND METHODS Bacterial luciferase was purified as previously described (18) from Photobacterium fischeri, Beneckea harveyt (19), and from dark mutants of B. harveyi, designated as M16 and M1S, which require exogenous aldehyde for bioluminescence. Enzyme concentrations were determined spectrophotometrically using a weight absorptivity of 0.94 (0.1%, 1 cm) at 280 nm, and a molecular weight of 79,000 (20). Lucifer, ase activity, expressed as peak intensity of emission, Io, was measured in a calibrated photometer (21) 1 gives circular dichroism (CD) spectra for free FMNH2, free luciferase, and an equimolar mixture of FMNH2 and luciferase, all three in the presence of dithionite.…”

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Bacterial luciferase requires one reduced flavin for light emission.

Becvar¹,

Hastings²

1975

Proc. Natl. Acad. Sci. U.S.A.

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Recent reports revive a hypothesis that the bacterial bioluminescence reaction involves two reduced flavin mononucleotide molecules per enzyme turnover. A twoflavin mechanism requires that the two flavins bind simultaneously or sequentially to the same or different sites on luciferase during a catalytic cycle. Measurements using equilibrium techniques show that the luciferase dimer has only a single reduced flavin binding site. (4) indicated that the bioluminescent reaction had a second order dependence on FMNH2 concentration, again suggesting the involvement of two flavins.Some years later, when the cad heterodimeric structure of luciferase was elucidated (5), the possibility that two reduced flavins might be required for activity could be envisioned in more specific terms. However, binding site determinations by a kinetic method showed that luciferase possesses only a single FMNH2 binding site per dimer (6). Furthermore, chemical modification studies (7,8) and mutant analyses (9) indicated that only the a subunit participates in the catalytic steps. A reaction mechanism involving the oxidation of a single FMNH2 and the concomitant oxidation of aldehyde was thus proposed (6, 10) and has been supported by a variety of experimental results since then (1, 11), including the demonstration that aldehyde is indeed oxidized to acid (12-15). However, based on the report that the quantum yield with respect to FMNH2 is approximately half that for aldehyde, it again was proposed that two reduced flavins are required per catalytic cycle (16,17 based on an absorptivity of 12,500 M-1 cm-1 at 445 nm (23). 2,2-Bis(hydroxymethyl)-2,2',2"-nitrilotriethanol (BisTris) and dithiothreitol were obtained from Sigma, decanal from Aldrich, bovine serum albumin from Pentex, dithionite (sodium hydrosulfite) from Mallinckrodt. Absorbance values were read with a Cary 15 spectrophotometer, circular dichroism spectra with a Jasco J-20 spectropolarimeter. Fig. 1 gives circular dichroism (CD) spectra for free FMNH2, free luciferase, and an equimolar mixture of FMNH2 and luciferase, all three in the presence of dithionite. The relatively large signal at 370 nm for the mixture is indicative of a complex between reduced flavin and luciferase. The stoichiometry of this complex was examined by the method of continuous variation of mole fraction according to job (24). When equimolar solutions of FMNH2 and luciferase are mixed in different proportions, the CD signal at 370 nm varies as a function of the relative mole fraction of the reactants (Fig. 2). Maximum signal develops for a mole fraction of flavin (and enzyme) near 0.5, indicating that luciferase has one binding site for FMNH2. Although these data are not ideal for the calculation of a dissociation constant Kd for the E:FMNH2 complex, they are consistent with the published value of 0.8 ,M (6). RESULTS AND DISCUSSION

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Structurally distinct bacterial luciferases

Cited by 152 publications

References 17 publications

Spectral detection of an intermediate preceding the excited state in the bacterial luciferase reaction

Spectral detection of an intermediate preceding the excited state in the bacterial luciferase reaction

Cloning and nucleotide sequences of lux genes and characterization of luciferase of Xenorhabdus luminescens from a human wound

Bacterial luciferase requires one reduced flavin for light emission.

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