3. ResultsTable 1. Activity and bioluminescent properties of the mutants studied.

Michael I. Koksharov, Natalia N. UgarovaDept. of Chemistry, Lomonosov Moscow State University, 119991, Moscow, Russiae-mail: mkoksharov@enz.chem.msu.ru

pH-tolerant mutants of Luciola mingrelica luciferasecreated by random mutagenesis

1. IntroductionMost firefly luciferases show a high pH-sensitivity of bioluminescence spectra which undergo a large shift from green to red light when lowering pH from 7.8 to 6.0. It is usually explained by the ability of luciferase to restrict the emitter – excited oxyluciferin – to exist in two different molecular forms. The exact mechanism of this process is still a matter of discussion.

A number of substitutions is known that lower the pH-sensitivity of luciferases. If combined they can even lead to pH-insensitive enzyme displaying only “green emitter” [1]. Such mutations are mainly localized in the region between 220 and 344 residues [2]. For 1-220 residues only two mutations (F16R and V182K in P. pyralis luciferase) were shown to affect bioluminescence spectra lowering red-shift at pH 6.5 [1].

We’ve conducted random mutagenesis of the first 225 residues of L. mingrelica luciferase to identify new substitutions that can change bioluminescence spectra.

Literature cited1. Law G, Gandelman O, Tisi L, Lowe C, Murray J. Mutagenesis of

solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH tolerance. Biochem J 2006;397:305-12.

AcknowledgmentsThis work was supported in part by the Russian Foundation for Basic Research (project No. 08-04-00624).

3. Resultsmutations Y35N, Y35H lead to pH-insensitive

bioluminescence spectra (pH 6.0–9.0)mutations Y35N, Y35H also prevent red-shift

at elevated temperatures: heating from 25˚C to 42˚C only broadens spectrum from 67 to 76 nm

mutation F16L significantly lowers the contribution of “red emitter” at low pH

double substitution F16L+A40S results in nearly pH-insensitive bioluminescence spectra probably due to cumulative effect of both mutations

A more detailed information about characterized mutants is given in Table 1.

Figure 1 illustrates the degree of pH-sensitivity of the wild-type luciferase and mutants of Y35 and F16.

Figure 1. Bioluminescence spectra of WT and mutant enzymes at different pH (25˚C) and at 42˚C (pH ~7,4).Buffer: 50 mM Tris-acetate, 10 mM MgSO4, 2 mM EDTA, 1.2 mM ATP, 0.3 mM luciferin.

610 (94)566 (75)130 ± 9S118C

λmax, nm(half-width)

566 (70)564 (67)20 ± 2F16L, A40S,Y11F, S118C

567 (90)610shoulder564 (70)60 ± 6F16L, I19T

564 (65)564 (67)60 ± 7Y35H, K191R564 (65)564 (67)70 ± 6Y35N610 (96)566 (76)100WTpH 6.1pH 7.8

Specific activity, %Mutations

2. MethodsMutant libraries were generated by error-prone PCR [3] with conditions leading to about 50% active clones in the library.

At pH 7.8 luciferase produces yellow-green light in vitro. But E. coli colonies expressing wild-type enzyme (WT) emit yellow-orange light, perhaps due to lowered intracellular pH causing an increase of the “red”component of the spectrum.

Plates with mutant colonies were screened by in vivobioluminescence. pH-tolerant mutants produce colonies retaining greenish color in contrast to WT as can be seen on a picture below.

4. ConclusionsMutation of Y35 appears to be the first known single substitution that makes bioluminescence spectrum of firefly luciferase pH-insensitive without appreciable contribution of the “red emitter” at pH 6-8.

Figure 2. Sequence alignment of several pH-sensitive and pH-insensitive luciferases. Key positions are shaded.

Judging from the sequence alignment (Figure 2), a change from aromatic to aliphatic residue in the 16th

position could be one of the factors leading to the pH-insensitivity of non-firefly luciferases since mutation of Phe16 to Leu (or Arg [1]) increase pH-tolerance.

Change from the large Tyr35 to a smaller histidine could also make a contribution to the pH-insensitivity of click-beetle luciferases.

In 2006 the crystal structures of L. cruciata luciferase in complex with reaction intermediate had been determined by Nakatsu et al. [4] for both WT enzyme and the red-emitting mutant S286N. This structures have only a few differences.

Figure 3. The structure of WT firefly luciferase [4] showing positions of mutations and the movement of the loop 233-237 in the “red-emitting” conformation.

Interestingly, one of the differences was the position of the loop 233-237 (Figure 3): in the WT enzyme P235 makes a close contact with Y35 but in the red-emitting mutant the loop displaces from Y35.

We can suppose that this close packing is necessary for the green-emitting conformation. Substitution of Y35 to a smaller residue could stabilize such packing making the usual conformational change at low pH unfavorable.

Y35 mutants can be useful in applications there only “green” emission is required independent of pH and temperature.

λ, nm

In vivo bioluminescence

Source of luciferase 11 16 19 22 35 40 pH-sensitive luciferases (from firefly beetles)

Luciola mingrelica VYGPLPFYPIEEG...QYAKL-GAIA...Lampyroidea maculata VYGPQPFYPIEKG...QYAQL-GAIA...Luciola cruciata VVGPKPFYPIEEG...RYAKL-GAIA...Photinus pyralis KKGPAPFYPLEDG...RYALVPGTIA...Lampyris noctiluca MHGPAPFYPLEDG...RYAQVPGTIA...Cratomorphus distinctus MYGPAPFSPLEEG...RYAQIPGTIA...Photuris pennsylvanica LIGPPPYYPLEEG...RYAAVPGTLA...Pyrocoelia miyako MHGHRHSILWEDG...RYAQVPGTIA...

pH-insensitive luciferases (from non-firefly beetles) Phrixothrix hirtus VNGDRPRDLVFPG...KYSYI--TDG...Phrixothrix vivianii RHGERPRDIVHPG...KFASF--PEA...Rhagophthalmus ohbai LHGAKPRDPLDLG...NFSFL--REA...Pyrearinus termitilluminans VYGPEPKHPLGNF...KHSHIP--QA...Pyrophorus plagiophthalamus VYGPEPLHPLEDL...KHSHLP--QA...

2. Viviani , Arnoldi F, Ogawa F, Brochetto-Braga M. Few substitu-tions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site-directed mutagenesis survey. Luminescence 2007;22:362-369.

3. Cirino P, Mayer K, Umeno D. Generating mutant libraries using error-prone PCR. Methods Mol Biol 2003;231:3-9.

4. Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H. Structural basis for the spectral difference in luciferase bioluminescence. Nature 2006;440:372-6.

500 525 550 575 600 625 6500,0

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500 525 550 575 600 625 6500,0

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Y35NY35H

pH 7.8

500 525 550 575 600 625 6500,0

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F16L

pH 7.8

pH 6.1

500 525 550 575 600 625 6500,0

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t=42°C

Y35N,H

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