Tài liệu Báo cáo Y học: Thermostability of manganese- and iron-superoxide dismutases from

Thermostability of manganese- and iron-superoxide dismutases

from Escherichia coli is determined by the characteristic position

of a glutamine residue

The´re` se Hunter1

, Joe V. Bannister1,2 and Gary J. Hunter1

1

Department of Physiology and Biochemistry, University of Malta, Msida, Malta; 2

Cranfield Biotechnology Centre, Institute of

BioScience and Technology, Cranfield University, Silsoe, Bedfordshire, UK

The structurally homologous mononuclear iron and man￾ganese superoxide dismutases (FeSOD and MnSOD,

respectively) contain a highly conserved glutamine residue in

the active site which projects toward the active-site metal

centre and participates in an extensive hydrogen bonding

network. The position of this residue is different for each

SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of

Escherichia coli). Although site-directed mutant enzymes

lacking this glutamine residue (FeSOD[Q69G] and

MnSOD[Q146A]) demonstrated a higher degree of selec￾tivity for their respective metal, they showed little or no

activity compared with wild types. FeSOD double mutants

(FeSOD[Q69G/A141Q]), which mimic the glutamine posi￾tion in MnSOD, elicited 25% the activity of wild-type

FeSOD while the activity of the corresponding MnSOD

double mutant (MnSOD[G77Q/Q146A]) increased to 150%

(relative to wild-type MnSOD). Both double mutants

showed reduced selectivity toward their metal. Differences

exhibited in the thermostability of SOD activity was most

obvious in the mutants that contained two glutamine resi￾dues (FeSOD[A141Q] and MnSOD[G77Q]), where the

MnSOD mutant was thermostable and the FeSOD mutant

was thermolabile. Significantly, the MnSOD double mutant

exhibited a thermal-inactivation profile similar to that of

wild-type FeSOD while that of the FeSOD double mutant

was similar to wild-type MnSOD. We conclude therefore

that the position of this glutamine residue contributes to

metal selectivity and is responsible for some of the different

physicochemical properties of these SODs, and in particular

their characteristic thermostability.

Keywords: superoxide dismutase; site-directed mutagenesis;

metal specificity; thermostability.

Iron superoxide dismutases (FeSOD, E.C.1.15.1.1) and

manganese superoxide dismutases (MnSOD) constitute a

class of structurally equivalent metalloenzymes prevalent in

prokaryotes and in eukaryotic mitochondria, respectively.

They exhibit a very high degree of homology in both

sequence and structure (Fig. 1). The SODs are active only in

dimeric association and all share structural homology in this

respect [1]. The metal cofactors are required to catalyse the

disproportionation of the superoxide radical into hydrogen

peroxide and molecular oxygen [2] in a cyclic, two-stage

oxidation-reduction mechanism:

M3þ þ O

2 ! M2þ þ O2 ð1Þ

M2þ þ O

2 þ 2Hþ ! M3þ þ H2O2 ð2Þ

where M represents either iron or manganese.

Selectivity of the proteins for their metal cofactor has been

demonstrated in vivo [3] and although apoenzymes of each

type of SOD may be reconstituted by the addition of metals,

the resulting enzyme is active only with the authentic metal at

its active centre [4–7]. A small number of cambialistic SODs

have been shown to be active with either iron or manganese,

though only those of Propionibacterium shermanii [8],

Bacteroides gingivalis [9] and Bacteroides fragilis [10]

demonstrate similar specific activities with either metal.

In all structures solved for the mononuclear SODs, the

metal ion is held in place by an absolutely conserved

quartet of residues comprising three histidines and one

aspartic acid which act as ligands to the metal (H26, H81,

D167 and H171 for Escherichia coli MnSOD, Fig. 1B.

This numbering will be used throughout except where

indicated) [11–21]. This conservation is also reflected in all

sequences elucidated for this large group of ubiquitous

enzymes. A fifth metal ligand, either water or hydroxide,

present in all structures produces a trigonal-pyramidal

geometry at the active site. A distorted-octahedral geo￾metry is assumed during catalytic turnover or inhibition,

Correspondence to G. J. Hunter, Department of Physiology and

Biochemistry, University of Malta, Msida, MSD 06, Malta.

Fax: + 356 21310577, Tel.: + 356 21316655,

E-mail: [email protected]

Abbreviations: Fe[Q69G], Fe[A141Q] and Fe[Q69G/A141Q],

Escherichia coli FeSOD mutated at glutamine 69 to glycine, alanine

141 to glutamine, or both, respectively; FeSOD, MnSOD, the iron- or

manganese-containing SOD, respectively; FXa, active form of

restriction protease factor X; GSH, reduced glutathione; GSt, gluta￾thione S-transferase; IPTG, isopropyl thio-b-D-galactoside;

Mn[G77Q], Mn[Q146A] and Mn[G77Q/Q146A], E. coli MnSOD

mutated at glycine 77 to glutamine, glutamine 146 to alanine, or both,

respectively; SOD, superoxide dismutase; wt, wild type.

Enzymes: iron superoxide dismutase from E. coli; SODF_ECOLI,

manganese superoxide dismutase from E. coli; SODM_ECOLI

(E.C. 1.15.1.1).

(Received 11 March 2002, revised 10 July 2002,

accepted 22 August 2002)

Eur. J. Biochem. 269, 5137–5148 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03200.x