Suadee, C

Nijivipakul, S

Svasti, J

Entsch, Barrie

Ballou, DP

Chaiyen, P

2007

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°C and pH 8. The measured Kd at 4°C for the binding of FMNH- to Lux_Vc was 1.8 μM whereas to Lux_Vh, it was 11 μM. 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°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.

Journal of Biochemistry, 142(4), p. 539-552

1756-2651

0021-924X

https://hdl.handle.net/1959.11/8613

en

Oxford University Press

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

Journal Article

Publication