Site-directed mutagenesis of bacterial luciferase: Analysis of the ‘essential’ thiol

It has been appreciated for many years that the luciferase from the luminous marine bacterium Vibrio harveyi has a highly reactive cysteinyl residue which is protected from alkylation by binding of flavin. Alkylation of the reactive thiol, which resides in a hydrophobic pocket, leads to inactivation of the enzyme. To determine conclusively whether the reactive thiol is required for the catalytic mechanism, we have constructed a mutant by oligonucleotide directed site-specific mutagenesis in which the reactive cysteinyl residue, which resides at position 106 of the α subunit, has been replaced with a seryl residue. The resulting α106Ser luciferase retains full activity in the bioluminescence reaction, although the mutant enzyme has a ca 100-fold increase in the FMNH₂ dissociation constant. The α106Ser luciferase is still inactivated by N-ethylmaleimide, albeit at about 1/10 the rate of the wild-type (α106Cys) enzyme, demonstrating the existence of a second, less reactive, cysteinyl residue that was obscured in the wild-type enzyme by the highly reactive cysteinyl residue at position α106. An α106Ala variant luciferase was also active, but the α106Val mutant enzyme was about 50-fold less active than the wild type. All three variants (Ser, Ala and Val) appeared to have somewhat reduced affinities for the aldehyde substrate, the valine mutant being the most affected. It is interesting to note that the α106 mutant luciferases are much less subject to aldehyde substrate inhibition than is the wild-type V. harveyi luciferase, suggesting that the molecular mechanism of aldehyde substrate inhibition involves the Cys at α106.