Basic study for the bioluminescence reaction on insect luciferases

甲虫、铁路虫荧光素酶(pH不敏感)和萤火虫荧光素酶(pH敏感)的比较表明，两组昆虫荧光素酶的一组保守残基不同，这可能与生物发光光谱的pH敏感性有关。在甲壳虫萤火虫荧光素酶中替换C258V导致红移突变(λmax=570 nm)。而Ohbai铁路虫荧光素酶(ROL)中V255C的替代对其无影响。Rol和Pyrearinus termitiluans(PYT)点击甲虫荧光素酶的绿光荧光素酶中的Thr226基因发生了红移(12~35 nm)，而陆地鞭毛虫(Phre)的红光荧光素酶中的T226N基因发生了10 nm的蓝移。在pML中，N230S突变为典型的红色突变体(λ=611 nm)。所有这些荧光素酶突变体的生物发光光谱都没有表现出与野生型荧光素酶相关的pH敏感性的变化，也没有明显的半带宽变化。总而言之，目前的数据表明，Thr226在两组甲虫荧光素酶中都是保持活性中心的重要残基。PH敏感型和pH不敏感型荧光素酶测定生物发光颜色的机理可能不同。

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.