Relationship between the conserved alpha subunit arginine 107 and effects of phosphate on the activity and stability of Vibrio harveyi luciferase.
The Arg107 of the alpha subunit is a conserved residue for all known bacterial luciferases. The phosphate moiety of the reduced flavin mononucleotide (FMNH(2)) side chain has been hypothesized to be anchored at this site (A. J. Fisher, F. M. Raushel, T. O. Baldwin, and I. Rayment Biochemistry 34, 6581-6586, 1995). Mutations of alphaArg107 of the Vibrio harveyi luciferase to alanine, serine, and glutamate were carried out to test such a hypothesis. These variants were characterized and compared with the wild-type luciferase with respect to their K(m) for decanal, FMNH(2), and reduced riboflavin in both low- (0.01 or 0.05 M) and high- (0.3 M) phosphate buffers at pH 7.0. Results are consistent with the hypothesized binding of the FMNH(2) phosphate group by alphaArg107. Moreover, the alphaArg107 residue was apparently important in the expression of the luciferase maximal activity and aldehyde binding. Phosphate ion is also known to have other effects on luciferase stability. We compared the three luciferase variants with the native enzyme with respect to the decay rate of the FMN 4a-hydroperoxide intermediate II, and rates of inactivation by trypsin digestion, modification by N-ethylmaleimide, and heat treatment in low- and high-phosphate buffers. On the basis of patterns of the phosphate effects, alphaArg107 appeared to be important to the enhancement of luciferase stability against trypsin proteolysis at high phosphate but was not involved in regulating the intermediate II decay or sensitivity to N-ethylmaleimide modification. Differential effects of mutations on luciferase thermal stability were observed. It is uncertain whether alphaArg107 is involved in the enhanced thermal stability of the native luciferase in high phosphate buffer.
Moore
C
C
Department of Biology, University of Houston, Houston, Texas, 77204-5513, USA.
Lei
B
B
Tu
S C
SC
D000409:Alanine; D000595:Amino Acid Sequence; D001120:Arginine; D017124:Conserved Sequence; D018698:Glutamic Acid; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism*; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D014733:Vibrio / Q000201:enzymology*
D046911:Macromolecular Substances; D010710:Phosphates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012694:Serine; D001120:Arginine; D008156:Luciferases; D000409:Alanine