Biochemical, catalytic, and spectroscopic studies of redox active enzymes; protein engineering
We
have recently established that catalytically essential diiron centers
are found in the plant stearoyl-acyl carrier protein Delta 9 desaturase
(Delta 9D) and the bacterial toluene-4-monooxygenase (T4MO). These
soluble, multicomponent enzymes utilize dioxygen and NADPH to catalyze
the oxidation of hydrocarbons. Delta 9D is ultimately responsible for
the biosynthesis of oleic acid, the most abundant unsaturated fatty
acid, while T4MO catalyzes the para-hydroxylation of toluene. These
enzyme complexes are of interest because they can oxidize stable C-H
bonds. In addition, we study flavoenzymes that initiate the bacterial
utilization of explosive compounds such as nitroglycerin and TNT as
nitrogen sources for growth. For all of these enzymes, we are
interested in determining the molecular details of the catalytic
reactions.
Broadly stated, our research goals are to
define the structure and the reactivity of the active site diiron
center, to probe the catalytic contributions of the active site protein
residues, and to determine the consequences of protein-protein and
protein-substrate interactions on the outcomes of enzymic catalysis.
Our research group makes extensive use of biochemical, catalytic, and
spectroscopic techniques as metalloenzyme active site probes. Through
application of these techniques, resting states as well as highly
reactive intermediates of the diiron enzyme catalytic cycle, are being
characterized. In addition to providing fundamental mechanistic and
structural information, these characterizations form the basis for
ongoing site-directed mutagenic manipulations of the protein- and
substrate-components of the enzyme complex. Since we obtain both of
Delta 9D and T4MO from recombinant overexpression systems, we also
remain interested in innovative ways to use advanced fermentation
technologies to further improve the productivity and yield of our
enzymes from these vectors.
It is reasonable to assume
that the catalytic diversity of the enzymes containing diiron centers
is related to the many possibilities for variation in the ligand types
and coordination numbers, in the geometry of ligand binding, and in the
polarity of the environment surrounding the diiron center. Highly
specific protein-protein interactions must also contribute to the rates
and yield of catalytic turnover. Through the detailed characterization
of the attributes of these versatile catalysts, we would ultimately
like to assemble bioengineered diiron enzymes capable of the oxidative
biotransformation of a wide variety of hydrocarbons.
For
the flavoenzymes, we are using a coordinated bacteriological,
biochemical, engineering, and structural approach to address the
problem of remediation of explosive compounds. This collaborative
effort involves studies from basic research to field application.