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3. ResultsTable 1. Activity and bioluminescent properties of themutants studied.

Michael I. Koksharov, Natalia N. Ugarova

Dept. of Chemistry, Lomonosov Moscow State University, 119991, Moscow, Russia

e-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 shiftfrom green to red light when lowering pH from 7.8 to6.0. It is usually explained by the ability of luciferase torestrict the emitter excited oxyluciferin to exist in twodifferent 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 evenlead to pH-insensitive enzyme displaying only greenemitter [1]. Such mutations are mainly localized in theregion between 220 and 344 residues [2]. For 1-220residues only two mutations (F16R and V182K in P.

pyralis luciferase) were shown to affect bioluminescence

spectra lowering red-shift at pH 6.5 [1].Weve conducted random mutagenesis of the first

225 residues of L. mingrelica luciferase to identify newsubstitutions 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 luciferaseimproves thermostability and pH tolerance. Biochem J2006;397:305-12.

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

3. Resultsmutations Y35N, Y35H lead to pH-insensitive

bioluminescence spectra (pH 6.09.0)

mutations Y35N, Y35H also prevent red-shiftat elevated temperatures: heating from 25

C to42

C only broadens spectrum from 67 to 76 nm

mutation F16L significantly lowers thecontribution of red emitter at low pH

double substitution F16L+A40S results innearly pH-insensitive bioluminescence spectra

probably due to cumulative effect of bothmutations

A more detailed information about characterizedmutants 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 mutantenzymes at different pH (25

C) and at 42

C (pH ~7,4).

Buffer: 50 mM Tris-acetate, 10 mM MgSO 4, 2 mM EDTA, 1.2mM ATP, 0.3 mM luciferin.

610 (94)566 (75)130 9S118C

max , nm

(half-width )

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

567 (90)610 shoulder

564 (70)60 6F16L , I19T

564 (65)564 (67 )60 7Y35H , K191R 564 (65)564 (67 )70 6Y35N610 (96 )566 (76 )100WT

pH 6.1 pH 7.8

Specificactivity, %Mutations

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

At pH 7.8 luciferase produces yellow-green light invitro . But E. coli colonies expressing wild-type enzyme(WT) emit yellow-orange light, perhaps due to loweredintracellular pH causing an increase of the redcomponent of the spectrum.

Plates with mutant colonies were screened by in vivo bioluminescence. pH-tolerant mutants produce coloniesretaining greenish color in contrast to WT as can be seenon a picture below.

4. ConclusionsMutation of Y35 appears to be the first known singlesubstitution that makes bioluminescence spectrum of firefly luciferase pH-insensitive without appreciablecontribution of the red emitter at pH 6-8.

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

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

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 histidinecould also make a contribution to the pH-insensitivity of click-beetle luciferases.

In 2006 the crystal structures of L. cruciata luciferasein complex with reaction intermediate had beendetermined by Nakatsu et al. [4] for both WT enzymeand the red-emitting mutant S286N. This structures haveonly a few differences.

Figure 3. The structure of WT firefly luciferase [4] showing

positions of mutations and the movement of the loop 233-237in the red-emitting conformation.

Interestingly, one of the differences was the positionof the loop 233-237 (Figure 3): in the WT enzyme P235makes a close contact with Y35 but in the red-emittingmutant the loop displaces from Y35.

We can suppose that this close packing is necessaryfor the green-emitting conformation. Substitution of Y35to a smaller residue could stabilize such packing makingthe usual conformational change at low pH unfavorable.

Y35 mutants can be useful in applications there onlygreen emission is required independent of pH andtemperature.

, nm

In vivo bioluminescence

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

Luciola mingrelica VYGPLP F YPIEEG...Q Y AKL-G A IA...Lampyroidea maculata VYGPQP F YPIEKG...Q Y AQL-G A IA...Luciola cruciata VVGPKP F YPIEEG...R Y AKL-G A IA...Photinus pyralis KKGPAP F YPLEDG...R Y ALVPG T IA...Lampyris noctiluca MHGPAP F YPLEDG...R Y AQVPG T IA...Cratomorphus distinctus MYGPAP F SPLEEG...R Y AQIPG T IA...Photuris pennsylvanica LIGPPP Y YPLEEG...R Y AAVPG T LA...Pyrocoelia miyako MHGHRH S ILWEDG...R Y AQVPG T IA...

pH-insensitive luciferases (from non-firefly beetles)Phrixothrix hirtus VNGDRP R DLVFPG...K Y SYI-- T DG...Phrixothrix vivianii RHGERP R DIVHPG...K F ASF-- P EA...Rhagophthalmus ohbai LHGAKP R DPLDLG...N F SFL-- R EA...Pyrearinus termitilluminans VYGPEP K HPLGNF...K H SHIP- - QA...Pyrophorus plagiophthalamus VYGPEP L HPLEDL...K H SHLP- - 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 usingerror-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 inluciferase bioluminescence. Nature 2006;440:372-6.

500 525 550 575 600 625 6500,0

0,5

1,0

WT

pH 7.8

pH 6.1

500 525 550 575 600 625 6500,0

0,5

1,0

pH 6.1

Y35NY35H

pH 7.8

500 525 550 575 600 625 6500,0

0,5

1,0

F16L

pH 7.8

pH 6.1

500 525 550 575 600 625 6500,0

0,5

1,0

t=42C

Y35N,H

WT

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