Basic study for the bioluminescence reaction on insect luciferases

通过比较萤火虫荧光素酶（pH敏感型）和甲虫和铁路蠕虫荧光素酶（pH不敏感型）的发光光谱，发现萤火虫荧光素酶（Pyrocoelia宫古（Pml））中存在一组与pH敏感性有关的保守残基，其中C258V的突变使荧光素酶的荧光光谱发生红移（λmax= 570 nm）。相比之下，在大白裂眼吸虫铁路蠕虫荧光素酶（Rol）中的取代V255C没有影响。在Rol和Pyrearinus termitilluminans（Pyt）点击甲虫荧光素酶的绿色发光荧光素酶中Thr 226的取代导致红移（12至35 nm），而在Phrixothrix hirtus（PhRE）铁路蠕虫的红色发光荧光素酶中T226 N的取代导致10 nm的蓝移。在PmL中，N230S突变产生一个典型的红色突变体（λ= 611nm）。所有这些荧光素酶突变体的生物发光光谱没有显示出改变的pH敏感性，也没有显着改变半带宽相对于野生型pH3酶。总之，目前的数据表明，Thr226是一个重要的残基保持活性中心的核心，在这两组的甲虫酶。pH敏感型和pH不敏感型生物发光酶的发光机理可能不同。

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.