Abstract
Enzyme function requires that enzyme structures be dynamic. Substrate
binding, product release, and transition state stabilization typically
involve different enzyme conformers. Furthermore, in multistep enzyme-catalyzed
reactions, more than one enzyme conformation may be important for
stabilizing different transition states. While X-ray crystallography
provides the most detailed structural information of any current methodology,
X-ray crystal structures of enzymes capture only those conformations
that fit into the crystal lattice, which may or may not be relevant
to function. Solution nuclear magnetic resonance (NMR) methods can
provide an alternative approach to characterizing enzymes under nonperturbing
and controllable conditions, allowing one to identify and localize
dynamic processes that are important to function. However, many enzymes
are too large for standard approaches to making sequential resonance
assignments, a critical first step in analyzing and interpreting the
wealth of information inherent in NMR spectra.
This Account
describes our long-standing NMR-based research into
structural and dynamic aspects of function in the cytochrome P450
monooxygenase superfamily. These heme-containing enzymes typically
catalyze the oxidation of unactivated C–H and C=C bonds
in a multitude of substrates, often with complete regio- and stereospecificity.
Over 600 000 genes in GenBank have been assigned to P450s,
yet all known P450 structures exhibit a highly conserved and unique
fold. This combination of functional and structural conservation with
a vast substrate clientele, each substrate having multiple possible
sites for oxidation, makes the P450s a unique target for understanding
the role of enzyme structure and dynamics in determining a particular
substrate–product combination. P450s are large by solution
NMR standards, requiring us to develop specialized approaches for
making sequential resonance assignments and interpreting the spectral
changes that occur as a function of changing conditions (e.g., oxidation
and spin state changes, ligand, substrate or effector binding). Solution
conformations are characterized by the fitting of residual dipolar
couplings (RDCs) measured for sequence-specifically assigned amide
N–H correlations to alignment tensors optimized in the course
of restrained molecular dynamics (MD) simulations. The conformational
ensembles obtained by such RDC-restrained simulations, which we call
“soft annealing”, are then tested by site-directed mutation
and spectroscopic and activity assays for relevance. These efforts
have gained us insights into cryptic conformational changes associated
with substrate and redox partner binding that were not suspected from
crystal structures, but were shown by subsequent work to be relevant
to function. Furthermore, it appears that many of these changes can
be generalized to P450s besides those that we have characterized,
providing guidance for enzyme engineering efforts. While past research
was primarily directed at the more tractable prokaryotic P450s, our
current efforts are aimed at medically relevant human enzymes, including
CYP17A1, CYP2D6, and CYP3A4.