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.
Madvar
Ali Riahi
AR
Department of Biochemistry, Faculty of Basic Sciences, Tarbiat Modarres University, Tehran, Iran.
Hosseinkhani
Saman
S
Khajeh
Khosro
K
Ranjbar
Bijan
B
Asoodeh
Ahmad
A
D000818:Animals; D002942:Circular Dichroism; D018698:Glutamic Acid / Q000378:metabolism*; D049453:Luciferases, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary
D018698:Glutamic Acid; D049453:Luciferases, Bacterial