Barynin, V. V.

Grebenko, A. I.

Revistes Catalanes amb Accés Obert

CRYSTALLIZATION OF CATALASES FOR
STRUCTURE INVESTIGATION
A.I. GREBENKO, V. V. BAR YNIN
Institute of Crystallography, USSR Academy of Sciences
Moscow 117333, USSR
Catalase (EC 1.11.1.6; H202:H202- oxidoreductase ) is an enzyme that
is present in cells of almost all living organisms . It decomposes hydrogen
peroxide to molecular oxygen and water:
2H202 - 2H20 + 02
The reaction consistis of two steps: the first step in the catalytic process
is the interaction of the first hydrogen peroxide molecule with enzyme. It
forms compound I, that is an oxidized form of the protein.
E + HOOH - HOH + EO (compound I)
At the second step compound I reacts further with the second hy-
drogen peroxide molecule, which reduces it to enzyme:
HOOH+EO-.HOH+E+02
Most biochemical and physico-chemical studies of catalase were done
with beef liver catalase [1 ]. Three-dimensional structure of beef liver catala-
se was investigated by r. Rossmann's group [2]. Less studied are catalases
from other sources such as plants, fungi and bacteria.
We crystallized the catalases from the fungus Penicillium vitale [3] and
two bacteria catalases Thermus thermophilus [4] and Micrococcus lysodeik-
ticus [5].
Molecular weights of all catalases are in region 250 - 300 kDa. Table 1
shows molecular weights and subunit composition of these catalases.
All catalases are oligomeric enzymes and consist of several subunits.
Molecules of Penicillium vitale catalase (PVC) and Micrococcus lysodeikti-
[Butll.Soc. Cat. Cien.],Vol.XII1,NUm .1,1992
368 1. 1, GRI BI\AO, 1.1. /i.IR)'.AI.A
Fig. 1. Crystalline precipitate at the bottom of an ultracentrifuge tube.
Fig. 2. The single crystals from ultracentrifuge precipitate (PVC-I).
[Butll.Soc.Cat .Cifn.],Vol.XIII, N6m.1,1992
CRIS .ILLIZ:1/7O.AOFC.IIAL.ISI:SFORSlRUCli'Rl^l.V1"1:S77G,1!lO.V 369
cus catalase ( MLC) are tetramers and each subunit of the molecule contains
one heme group with an iron atom in the active site.
Catalase from bacteria Thermus thermophilus (TTC) is non-heme Ca-
talase. It consists of six subunits and in the active site contains two manga-
nese atoms.
Sources of isolation of these three catalases were the protein suspen-
sions . After several stages of extraction , precipitation and purification by
the biochemical methods , we have got the homogeneous solutions of catala-
ses, which were suitable for crystallization.
Table 1. Molecular weights, subunit number of catalases.
Protein
Molecular
weight
Numberof
subunits
Metal in
active site
Catalase from
Penicillium vitale 290,000 4 Fe
Catalase from
Thermos thermophilus 210,000 6 2 Mn
Catalase from
Ificrococ(ush'sodeikticus 232,000 4 Fe
Table 2. Crystallization of Penicillium vitale Catalase (PVC), Thermus thermophilus Cata-
lase (TTC) and Micrococcus lysodeikticus Catalase (MLC)
Concentration Size of Growth
Protein of protein pH Buffer Precipitant
stalscr time
mg/ml y
0.1M MPD
PVC 0.15-020 5.5 Sodium- 15-18`%% 1.Ox1.5mm 180 hours
I acetate
(at 4°C
0.1 M
PVC 18.0-24.0 5.5 Sodium- (NH,),SOa 0.5-0.7 min 4-7 days
II acetate 1.4M
0.05M
TTC 25.0-30.0 6.0 Potassium (NH4)2SO4 1.0-1.5mm 4-7davs
phosphate 1.5M
0.05M
MLC 20.0-25.0 5.2 Sodium- (NH4)2SO4 0.5x1.2mm 4-7days
acetate 1.2M
[ ButllSoc.Cat.Cien.],Vol.xIII,Num.I,I992
1 /. GR/h/\/^U. 1 1 R V<>\l\
F i,4. 3. Scheme o I , i ^ ^ta I I i /,it ion cc I I.
i^;. d. Crvscsls of Penicilliurn z^itale ca^alase (PVC-II).
^13udlSuc.Cat.Cien.],b'^il.XIII,[^iimJ,l991
CRYSl'ALLlL;1'l'lU:'01 CA/A1 1SL.SFORSIR! (7[X1 l\\iS'1101ll0\ 371
7
Fig. 5. Crystals of Thermos thermophilus catalase.
L
Fig. 6. Crystals of Micrococcus lysodetkttcus catalase.
lButll.Soc.(. .(icn .], Vol.XIII,Num.I,1992
7 2 l.l. (,R1 81.AAO, A.1. B.IR)A/.A
Table 3. Parameters ofcatalase crystals.
Catalase of Mol. mass
in kDa Space group
Unit cell
in A
Mol. in
asv'mm. unit
X-ray
resol. in A
Penicillium 290 P3,21 a = 144.4 1 /2 2.0
vitale (4 x 72) c = 133.8
Therrnus 210 P213 a = 133.4 I'll 3.0
thermophllus (6 x 35)
Aficrococcuc 232 P4,2i2 a = 106.7 1/4 2.5
lysocleikncus (4 x 58) c = 106.3
We used different methods for crystallization of the proteins. For crvs-
tallizatioin of PVC-I we used a rather unusual method, method of crystalli-
zation in an ultracentrifuge [6[.
Test tubes with the protein solution were rotated in a Beckman ultra-
centritut;e at 15 000 rev./min for 180 hours, at 4°C. Crystals of PVC-I as can
be seen from Table 2 were grown from protein solution, containing 15-18%
2-methyl-2,4-pcntandiol in Sodium acetate buffer.
Crystalline precipitate of irregular shape crystals was at the bottom of
the centrifuge tube (figure 1). Crystals reached a size of 1.5 mm and several
single crystals were separated easily from precipitate (figure 2).
For crystallization of PVC-I1, '1 1'C and MLC we used a more com-
mon method of crystallization of proteins: vapour diffusion method at
room temperature in a special cell (See figure 3). Crystals of these proteins
were grown at conditions specified in 'f'able 2. Crystals are shown in figures
4-6.
All catalase crystals had a good quality and were investigated by X-ray
analysis. Parameters of the crystals are listed in Table 3. Quantity of crystals
was good for X-ray investigation of three-dimensional structure of catalase
molecules of different organisms [3, 5, 7].
A itsin CC
Catalases from three microorganizrns, the fungus Penicillimsr :,itale and two bac-
teria Thermos therntophilus and tlicrocnccns Ir ocleikticus were crvstalliied for X-ray
structure investigation. The suitable crystals were grown by ultracentrifuge technique
and vapour diffusion hanging drop technique using 2-methyl 2,4-pentandiol and ail
monium sulphate as the precipitants.
ButII.S(1c.Cu.(:im ],yoI.AIII,yum.1,1992
CKY;S7 ALLILA170A 01 CAlALA.SLS FORSIRUC;7 UKE LV1L.S77G;1770.A 373
REFERENCES
1. ScHON[?,AUM, G.R. and CIIANCI, B. (1976) The Enzymes, v. 13, p. 363-408,
Acad. Press, New York.
2. MURTHY, M.R.N., RI?ID, T.J. 111, SICIGNANO, A., TANAKA, N. and Koss-
MANN, M.G. (1981) J. Mol. Biol. v. 152, p. 465-499.
3. VAINSHTI{IN, B.K., MIILIK-ADAMYAN, W.R., BAKYNIN, V.V., VAGIN, A.A.,
GKh:BI:NKO, AT., Bolusov, V.V., BAKTLLS, K.S., FITA, I. and RossMANN,
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5. Yusii ov, E.F., GIU:BLNKO, A.I., BARYNIN, V.V., MuKSHuDov, G.N., VA-
GIN, A.A., MI :LIK-ADAMYAN, W.R. and VAINSHTFIN, B.K. (1989) Kristallo-
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ButII.Soc ( aC .(.1CU.],VoI.XI11, Nun1.1,1992


Crystallization of catalases for structure investigation

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