The major
thrust of our research is to elucidate the molecular and genetic basis of
sterol biosynthetic reactions and to elucidate the role sterol structure
plays in biological activity and evolution. Our goal is to establish a full
understanding of sterol diversity as a prerequisite for the rational design
of medicinal drugs, antifungal and antiinsect compounds to control the
production and processing of sterol synthesis in normal and diseased
systems. We also are researching phytosterol homeostasis to generate
transgenic plants with modified sterol compositions that possess
value-added traits.
For example, one area of interest is
the study of a key enzyme in ergosterol synthesis responsible for the
critical slow step controlling the transmethylation reaction at C-24 in the
fungal sterol side chain, the sterol methyl transferase (SMT). The C-24
methyl group is an essential feature to the role of sterols in fungal
physiology. Animals do not synthesize 24-alkyl sterols. Differences in
sterol structure between animals and fungi (particularly opportunistic
pathogens) provide a unique opportunity to develop rational control of
disease through differences in sterol synthesis. Thus, we are attempting to
understand the catalytic mechanism and structure of SMTs from different
sources. Specifically, we have cloned, sequenced and generated large amounts
of pure recombinant SMT to study its catalysis. Using a mechanism-based
approach we have designed a set of substrate, transition state and product
analogs which to inhibit enzyme activity. It appears that some of these
compounds have the potential to serve as taxa-specific inhibitors with high
specific activity.
A few other specific systems that are
under investigation are the human sterol 8-to-7 isomerase (in cooperation
with AstraZeneca Pharmaceutical), which we have overexpressed in bacteria
and yeast and the 14a-demethylase
enzyme from Mycobacterium tuberculosis (in cooperation with Michael
Waterman at Vanderbilt Medical School) which we have overexpressed in
bacteria. In both cases, we have studied the pure enzyme kinetically. We
have also generated transgenic plants with modified phytosterol compositions
(in cooperation with Henry Nguyen at Texas Tech University). We have
introduced foreign SMT genes from yeast into tobacco and tomato plants and
utilized antisense technology to impair SMT activity in these plants.
Finally, we have determined that the classic acetate/mevalonate pathway to
ergosterol is not operational in the yeast-like Prototheca wickerhamii
which synthesizes its sterol from the mevalonate-independent pathway.
Students, Post-doctoral Research Associates and Visiting Scientists get
training in my laboratory in organic synthesis, analysis, biochemistry,
enzymology, molecular biology and microbiology. The techniques and
experimental approaches developed in this laboratory are expected to be
broadly applicable to the understanding of enzyme mechanistic and
biosynthetic investigations.
