X-ray Diffraction and Computer Simulation Studies of Protein Function 
tion of 2 A using synchrotron x-ray data, shows 
that despite these differences the tertiary struc- 
ture of TR is closely similar to that of GR. How- 
ever, the quaternary structures and active-site ar- 
chitectures are unrelated, and the two enzymes 
appear to have acquired the disulfide reductase 
activity independently. These results provide a 
striking example of modular evolution of three- 
dimensional protein structure and enzyme 
function. 
Trypanothione reductase (TrypR) represents 
an enzyme target for drug intervention in African 
trypanosomiasis, Chagas' disease, and leishman- 
iasis. The protozoan parasites do not possess GR, 
and instead use a glutathione-based peptide, try- 
panothione, for the essential reduction of gluta- 
thione. Trypanothione is in turn reduced by 
TrypR, which was first characterized from an in- 
sect trypanosomatid, Crithidia fasciculata. This 
analysis revealed that it is very similar to GR in 
size, catalytic mechanism, and active-site 
structure. 
The two enzymes, however, are mutually ex- 
clusive for substrate. This, combined with the 
known susceptibility of trypanosomatids to oxi- 
dative stress in the absence of reduced glutathi- 
one, makes TrypR a promising target for the devel- 
opment of therapeutic agents that are not toxic to 
the host cell. We have obtained nicely diffracting 
single crystals of trypanothione reductase from C. 
fasciculata and have recently solved the struc- 
ture to 2.4 A resolution by molecular replace- 
ment, using the structure of human erythrocyte 
GR as a search model (in collaboration with 
Anthony Cerami) . We are in the process of identi- 
fying the residues that are responsible for the 1 0^ 
enhancement in the turnover of glutathione for 
trypanothione in the parasite enzyme. 
An extremely interesting and well-character- 
ized redox-based regulatory system is the bacte- 
rial response to oxidative stress. Exposure of E. 
colt or Salmonella typhimurium to low levels of 
hydrogen peroxide results in subsequent resis- 
tance to high levels. This is due to the induction 
of 30 proteins. The OxyR protein is responsible 
for the peroxide induction of nine of these, in- 
cluding GR, catalase, and an alkyl hydroperoxide 
reductase that is similar in sequence to TR. 
Unlike many bacterial regulatory systems, 
where distinct proteins are involved in sensing 
the environmental change and in activating tran- 
scription, OxyR is both the sensor and the tran- 
scriptional activator. The levels of OxyR do not 
change significantly when the cell is challenged 
with peroxide. Rather, OxyR undergoes rapid 
and reversible changes in its DNA-binding proper- 
ties. Depending on which site it is bound to, 
OxyR acts as either an activator or a repressor of 
transcription. We have entered into a collabora- 
tion with Gisela Storz to crystallize OxyR and de- 
termine its three-dimensional structure, its mode 
of binding to DNA, and the mechanism of its re- 
sponse to oxidative stress. Since OxyR is member 
of a large family of bacterial regulatory proteins 
of as yet unknown structure or mechanism, our 
studies are likely to have broad implications. 
254 
