Three-Dimensional Structures of Biological Macromolecules 
ing a crosslinked pair of thymine bases. These ex- 
periments have to be done in the dark or under 
yellow or red light, to prevent the enzyme from 
repairing and releasing the substrate. 
Because light is an essential ingredient of the 
enzymatic reaction of DNA photolyase, the en- 
zyme-substrate complex will be a suitable system 
to study the time course of the reaction, using the 
Laue technique. This technique employs the 
broad spectrum of x-ray wavelengths in the 
synchrotron's powerful beam to record within a 
very short time a sufficient fraction of a crystal's 
dilfraction pattern for structural interpretation. 
In such an experiment the crystal is irradiated 
with a light pulse suitable to trigger the enzy- 
matic reaction, and diffraction patterns are re- 
corded at different times after the pulse. These 
data can provide snapshots of the structural rear- 
rangements during the reaction and thus contrib- 
ute to an understanding of the enzyme's 
mechanism. 
Cytochrome P-450 Enzymes 
P-450 enzymes are members of a superfamily 
of b-type heme proteins that catalyze hydroxyla- 
tion of organic substrates using electrons from 
reduced nicotinamide adenine dinucleotide 
phosphate (NADPH) and molecular oxygen. 
They consist of a polypeptide chain of about 420 
amino acids and a heme group as part of their 
active site. P-450s occur in organisms as different 
as bacteria and humans. Some of them play spe- 
cific roles in biosynthetic pathways, such as for- 
mation of glucocorticoids, androgens, and estro- 
gens from cholesterol; others are involved in 
detoxification of foreign substances by making 
them more soluble and so facilitating their excre- 
tion. However, in some cases hydroxylation of 
foreign substances creates highly efficient carcin- 
ogens; thus the effects of P-450s are not always 
beneficial. 
P-450s occur in two classes, distinguished by 
the way in which electrons from NADPH are 
transferred to the heme group. Class I P-450s re- 
ceive the electrons via a flavoprotein and an iron- 
sulfur protein; class H enzymes are reduced di- 
rectly by a protein containing the cofactors flavin 
mononucleotide and FAD. The three-dimensional 
structure of only one class I P-450, the camphor 
hydroxylase P-450cam' is known at atomic 
resolution. 
To learn more about the structural variation 
within the cytochrome P-450 family, about dif- 
ferences between class I and class II enzymes, and 
about the spectrum of reactions catalyzed by 
P-450s, we collaborate with Julian Peterson and 
his colleagues (University of Texas Southwestern 
Medical Center at Dallas) to analyze the three- 
dimensional structures of a class I and a class II 
enzyme. The class I P-450 was isolated from 
Pseudomonas; it hydroxylates the hydrocarbon 
a-terpineol and was therefore named P-450,p^. 
The class II P-450 is the cytochrome part of an 
enzyme from Bacillus megaterium that is 
unique in combining a P-450 domain and a P-450 
reductase domain in a single polypeptide chain 
of 1,066 amino acids. The cytochrome domain, 
P-450bm-3. consists of the 471 amino-terminal 
amino acids; it was cloned and expressed at high 
levels in E. coli. Its functions include hydroxyla- 
tion and epoxidation of long-chain fatty acids. 
Well-ordered crystals of both P-450s have been 
grown. At CHESS, crystals of P-450t^rp diffracted 
to about 2.0 -A resolution, and crystals of 
P-450bm-3 diffracted to better than 1.5 -A resolu- 
tion. Molecular replacement calculations, using 
the P-450cam model, produced a plausible solu- 
tion for the crystal structure of P-450terp- Multiple 
isomorphous replacement with three heavy-atom 
derivatives allowed the calculation of an electron 
density map at 3-A resolution for P-450bm.3; this 
map is currently being interpreted. Model build- 
ing and crystallographic refinement will result in 
accurate models of both proteins in the near fu- 
ture. We also plan to study substrate binding and 
structures of site-specific mutants. 
Other Projects 
In addition to the projects described above, we 
also work on the determination of the three- 
dimensional structures of several other proteins, 
including the catalytic domain of human HMG- 
CoA reductase, a key enzyme in the synthesis of 
cholesterol and a likely target for drugs; human 
synapsin I, a protein binding to synaptic vesicles 
and mediating their release; the small GDP- 
binding protein smgp25A from bovine brain; the 
DNA-binding protein myogenin; the SecA protein 
from E. coli, one of the key parts of the protein 
export system; mammalian phosphofructokinase; 
and ribonuclease inhibitor from pig liver. 
The work on cytochrome b/Cj complexes and 
on DNA photolyase is also supported by a grant 
from the Welch Foundation. 
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