Basic study for the bioluminescence reaction on insect luciferases

通过比较click beetle和railroadworm的荧光素酶（pH-insensitive）和firefly的荧光素酶（pH-sensitive），发现两组荧光素酶之间存在一组不同的保守残基，这些残基可能与生物发光光谱的pH敏感性有关。在miyako （Pml）萤火虫荧光素酶中替换C258V导致突变体红移（λmax= 570 nm）。而取代物V255C对铁蚕荧光素酶（Rol）无影响。用Thr226替代Rol和Pyrearinus termitilluminans （Pyt）点击虫的绿光荧光素酶可产生12 ~ 35 nm的红移，而用T226N替代Phrixothrix hirtus （PhRE）铁路虫的红光荧光素酶可产生10 nm的蓝移。在PmL中，替换N230S得到一个典型的红色突变体（λ= 611nm）。与野生型荧光素酶相比，所有这些荧光素酶突变体的生物发光光谱没有显示出ph敏感性的改变，也没有显着改变半带宽。总的来说，目前的数据表明，Thr226是保持两组甲虫荧光素酶活性位点核心的重要残基。ph敏感型荧光素酶和ph不敏感型荧光素酶测定生物发光颜色的机制可能不同。

The comparison of click beetle and railroadworm luciferases (pH-insensitive) with firefly luciferases (pH-sensitive) showed a set of conserved residues differing between the two groups which could be involved with the bioluminescence spectra pH sensitivity.The substitution C258V in Pyrocoelia miyako (Pml) firefly luciferase resulted ina red-shifted mutant (λmax= 570 nm). In contrast, the substitution V255C in Ragophthalmus ohbai railroad worm luciferase (Rol) had no effect. Substitution of Thr226 in the green light emitting luciferases of Rol and Pyrearinus termitilluminans (Pyt) click beetle luciferases resulted in red-shifts (12 to 35 nm), whereas the substitution T226N in the red light emitting luciferase of Phrixothrix hirtus (PhRE) railroadworm resulted in a 10 nm blue-shift. In PmL the substitution N230S resulted in a typical red mutant (λ= 611nm). The bioluminescence spectrum of all these luciferase mutants did not showed altered pH-sensitivity nor considerably changed half-bandwidth in relation to the wild-type luciferases. Altogether present data suggest that Thr226 is an important residue for keeping active-site core in both groups of beetle luciferases. The mechanism for bioluminescence color determination between pH-sensitive and pH-insensitive luciferases could be different.