ИСТИНА

Insufficient thermostability of wild-type (WT) firefly luciferases often limits their application. The substitution A217L is known to greatly increase thermal stability of many firefly luciferases, for example, Luciola lateralis (Lll), Luciola cruciata (Lcl) and Photinus pyralis (Ppl) luciferases [1]. However, for Hotaria parvula firefly luciferase (Hpl), which shares 98% sequence identity with Luciola mingrelica luciferase (Lml), the A217L mutation is known to dramatically decrease catalytic activity more than 1000-fold [1]. We have analyzed the environment of A217 in the 3D-structure of Lml and compared it with that in Hpl, Lll, Lcl, Ppl in order to propose possible additional mutations that would retain the high thermal stability of the mutant A217L while preserving the high level of activity. The 7Å environment of A217 is identical in Lml and Hpl, thus it is safe to assume that in both these highly homologous enzymes the single substitution A217L would lead to the loss of activity. The neighboring residue G216 and the more remote S398 appeared to be the key positions that distinguish the environment of A217 in a small subgroup of luciferases including Lml and Hpl from that of Lll, Lcl, Ppl and most others. In other beetle luciferases the position 216 is occupied with a residue having a side group (in contrast to G216 in Lml) and the position 398 is generally occupied with methionine. We decided to eliminate these differences to make the A217 environment similar to that of Lcl and thus possibly prevent the loss of activity in the case of the substitution A217L in Lml. The double mutant G216N/A217L had a half-life of 160 and 80 min at 42°C and 45°C, respectively, which is 18- and 28-fold increase in stability over WT Lml. However, it retained only 10% of activity. The loss in activity was accompanied by a large red shift of the bioluminescence emission maximum from 566 to 611 nm compared with the wild type enzyme. This shift was caused by the significant increase of the contribution of the “red emitter” in the bimodal spectrum of firefly luciferase (Fig. 1). Interestingly, the red shift of this mutant and its bioluminescence spectra were similar to that of the mutant H433Y studied previously [2], which is located 23 Å away from the position 217. Since the change G216N was insufficient to obtain a fully active luciferase, the additional substitution S398M was introduced. The mutant S398M alone showed catalytic properties and stability similar to that of WT. Its bioluminescence spectra were slightly less pH- and temperature sensitive. The triple mutant G216N/A217L/S398M possessed the high thermal stability of the double mutant as well as high activity and yellow-green bioluminescence of the wild-type enzyme [3]. Thus, the substitution S398M was able to effectively restore the activity and color of the mutant G216N/A217L (Fig. 1, Fig. 2). Figure 1. Bioluminescence spectra of WT (1) luciferase and the mutants S398M (2), G216N/A217L (3), G216N/A217L/S398M (4), H433Y (5) at pH 7.8 and pH 6.0 (25°C). Figure 2. In vivo bioluminescence of E. coli colonies producing WT (1) luciferase and the mutants S398M (2), G216N/A217L (3), G216N/A217L/S398M (4). In conclusion it can be stated that rational protein design of a residue microenvironment can be an effective strategy when a single mutation does not lead to the desirable effect reported for the similar substitution in a homologous enzyme. REFERENCES 1. Kitayama, A., Yoshizaki, H., Ohmiya, Y., Ueda, H. and Nagamune, T. Creation of a thermostable firefly luciferase with pH-insensitive luminescent color. Photochem. Photobiol. 2003; 77: 333-338. 2. Ugarova, N., Maloshenok, L., Uporov, I. and Koksharov, M. Bioluminescence spectra of native and mutant firefly luciferases as a function of pH. Biochemistry (Moscow). 2005; 70: 1262-1267. 3. Koksharov, M. I. and Ugarova, N. N. Triple substitution G216N/A217L/S398M leads to the active and thermostable Luciola mingrelica firefly luciferase. Photochem. Photobiol. Sci. 2011; 10: 931-938.