Bacterial luciferase activity does not require a disulfide-dithiol conversion

Tu, Shiao‐Chun; Baldwin, Thomas O.; Becvar, James E.; Hastings, J. Woodland

doi:10.1016/0003-9861(77)90120-5

Cited by 21 publications

(7 citation statements)

References 29 publications

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“…mediate II of both P.fischeri (-) and B. harveyi (-). Spectra of intermediate I were obtained as described previously (Tu et al, 1977). Spectra of intermediate II were determined by an extrapolation to zero time of the data from experiments of Figures 2 and 3.…”

Section: Resultsmentioning

confidence: 99%

“…The luciferase preparations were judged to be greater than 95% homogeneous bydisc gel and sodium dodecyl sulfate gel electrophoresis. Enzyme concentration was determined spectrophotometrically using a weight absorptivity of 1.2 (0.1%, 1 cm) at 278 nm (Tu et al, 1977) and a molecular weight of 79 000, although recent results in this laboratory indicate the actual molecular weight may be slightly (~5%) lower. Luciferase activity was measured in a calibrated photometer (Mitchell & Hastings, 1971;Hastings & Weber, 1963) by injection of 1 mL of 5 X 10-5 M FMNHi (catalytically reduced by Ha/Pt asbestos) into 1 mL of assay buffer (0.02 M phosphate, pH 7, 0.2% bovine serum albumin, and the optimal amount of a 0.1% (v/v) sonicate of decanal in water) containing enzymes.…”

Section: Methodsmentioning

confidence: 99%

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Activity and stability of the luciferase-flavin intermediate

Becvar¹,

Tu²,

Hastings³

1978

Biochemistry

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A luciferase intermediate in the bacterial bioluminescence system, which is formed by reaction of enzyme with reduced flavin mononucleotide (FMNH2) and oxygen, is shown to emit light with added aldehyde under anaerobic conditions. The reaction with oxygen is thus effectively irreversible under the conditions used. The flavin chromophore has an absorption maximum at about 370 nm and the potential activity (bioluminescence yield) in the further reaction of the isolated intermediate with aldehyde is strictly proportional to the amount of this flavin chromophore.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Activity and stability of the luciferase-flavin intermediate

Becvar¹,

Tu²,

Hastings³

1978

Biochemistry

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“…Hastings, 1963). This model was subsequently abandoned when (1) it became evident that the enzyme contained no disulfide linkages (Hastings, 1968;Tu et al, 1977) and ( 2) the original observation that FMNH2 was oxidized to FMN in the absence of oxygen proved irreproducible (Gibson et al, 1966;Hastings & Gibson, 1967). Later work demonstrated a single FMNH2 binding site per molecule of enzyme (Meighen & Hastings, 1971) and invalidated (Becvar & Hastings, 1975) the hypothesis that two flavins participated in the bioluminescence reaction.…”

Section: Discussionmentioning

confidence: 99%

Reversible inhibition of the bacterial luciferase catalyzed bioluminescence reaction by aldehyde substrate: kinetic mechanism and ligand effects

Holzman¹,

Baldwin²

1983

Biochemistry

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after reduction to 2-hydroxybutyrate with lactate dehydrogenase. These experiments and additional experiments on the stereospecificity of pyruvate kinase with different PEP analogues will be published separately. AcknowledgmentsWe gratefully acknowledge the help of Klaas Dijkstra with the NMR spectrometer, and we thank Tom Duffy for preparing pure E and Z isomers of PEB.ABSTRACT: The bioluminescence reaction catalyzed by bacterial luciferase from the luminous marine bacterium Vi'ibrio harveyi was found to be subject to reversible, chain length dependent inhibition by aldehyde substrate. The stoichiometry of aldehyde to luciferase in the bioluminescence reaction was 1 :l; the kinetics of substrate inhibition were consistent with the binding of a second molecule of aldehyde to luciferase to form an enzymatically inactive complex. These findings indicated that aldehyde interactions with bacterial luciferase from V. harueyi could not be adequately described by simple Michaelis-Menten kinetics. The binding of n-decanal to luciferase and the bioluminescence reaction velocity were dependent on buffer composition and concentration. Phosphate

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“…Luciferase was purified according to the procedure of Holzman and Baldwin (1982), and was >95% pure as judged from SDS-gel electrophoresis. The concentration of the enzyme was estimated using a molecular weight of 79kDa and an E,,, value of 74000 M-I cm-' (Tu et al, 1977). Absorbance spectra were recorded with a Kontron Uvikon 820 spectrophotometer modified for the simultaneous determination of light emission, as described earlier (Kurfurst et al, 1984).…”

Section: Methodsmentioning

confidence: 99%

Boronic Acids as Mechanistic Probes for the Bacterial Luciferase Reaction

Ahrens

Macheroux

Eberhard

et al. 1991

Photochem & Photobiology

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Abstract-Bacterial luciferase uses long chain aldehydes as substrates. Alkylboronic acid analogs of these substrates with chain lengths of C 7 and C. have been synthesized, characterized, and used as mechanistic probes for the light emitting reaction. They behave as inhibitors in the in vitro luminescence reaction. Contrary to an earlier report (Macheroux and Ghisla, 1985, Nachr. Chem. Tech. Lab. 33, 785-790) they are not substrates for bacterialluciferase, in that they do not lead to light emission and are not oxidized by the flavin-4a-hydroperoxide to the products boric acid and the corresponding alcohol, as would be expected from a Baeyer-Villiger reaction. However, the particular conformation of a putative boronic acid hydroperoxide at the active center might be such that it would preclude a Baeyer-Villiger fragmentation. Thus, while the results do not support the postulate that luciferase proceeds via a Baeyer-Villiger mechanism, they also do not exclude it. A further observation was that the endogenous light emission (no added aldehyde) decays more rapidly than does the luciferase bound flavin-4a-hydroperoxide. This suggests that the endogenous light is not caused by the decomposition of the flavin-4a-hydroperoxide.An adaptation of the Baeyer-Villiger mechanism L-FMNH-4a-OOH + R-CHO (cf. Scheme 1) was proposed by Eberhard and Hastings (1972) to explain this process, and has been frequently quoted in textbooks and reviews as a viable path (Branchaud and Walsh, 1985). However, this mechanism suffers from the lack of a rationale for the generation of an excited state. It has been demonstrated that the flavoprotein cyclohexanone monooxygenase is able to process alkyland arylboronic acids as substrates, and good arguments have been put forward for this reaction proceeding via a Baeyer-Villiger type of mechanism (Branchaud and Walsh, 1985;Walsh and Chen, 1988). Furthermore, the oxidation of boronic acid substrate analogs to the corresponding boric acid

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Bacterial luciferase activity does not require a disulfide-dithiol conversion

Cited by 21 publications

References 29 publications

Activity and stability of the luciferase-flavin intermediate

Activity and stability of the luciferase-flavin intermediate

Reversible inhibition of the bacterial luciferase catalyzed bioluminescence reaction by aldehyde substrate: kinetic mechanism and ligand effects

Boronic Acids as Mechanistic Probes for the Bacterial Luciferase Reaction

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