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Tweaking Taxol points way to a greener, more productive future 3/24/2006
Our
research uses a multidisciplinary approach to elucidate
biosynthetic pathways of secondary metabolites that have a
potentially beneficial biological effect. Acquisition and
characterization of the genes, and corresponding gene
products, on various pathways to bioactive compounds provides
the field of natural products biochemistry with tools for
potential application in the biotransformation of natural
products or synthetically-derived chemicals that are
non-native substrates of the isolated enzymes. These
functionally-defined genes can also be bioengineered into a
suitable host organism to potentially alter the metabolic
profile of interesting molecules in vivo
Kevin Walker, a chemistry and biochemistry and molecular biology assistant professor, in the March 24 issue of Chemistry & Biology, reports a step toward manufacturing more-potent Taxol molecules that could potentially reduce treatment dosages. The methods described minimize dangerous chemical usage, and put E. coli to work in the production process. “We’re trying to develop a biosynthetic process for the drugs that circumvents the use of organic solvent-based methods requiring costly waste management,” Walker said. “This attempt is a green chemistry approach to produce more potent versions of Taxol.” Taxol – generically known as paclitaxel – is a top-selling cancer-fighting drug. It’s most commonly used against ovarian and breast cancers, but currently is used in certain aspects of heart disease treatment, and is showing promise in Alzheimer’s therapy. Taxol is derived in small quantities from the Pacific yew tree. To fulfill large-scale production, pharmaceutical companies isolate, from the tree, an abundant natural product that is synthetically converted to Taxol in the laboratory. Now, as abundant molecules from the yew are being synthetically modified for new, more potent versions of Taxol, Walker, along with Catherine Loncaric, a visiting research associate, and undergraduate Erin Merriweather, is looking for alternative, biological routes to introduce the modifications. Walker’s laboratory makes use of recently identified genes of the yew that produce enzymes that craft the pathway to Taxol. The targets: five enzymes that biosynthetically decorate the core of the Taxol molecule. The enzymes in natural and, potentially, genetically modified form can be used to produce second-generation versions of the drug. Walker said the added advantage is that water-based chemicals rather than chlorinated solvents can be used with his methods. For reasons ranging from competitive advantage to corporate culture, or to a desire to be a good corporate citizen, pharmaceutical companies are drawn to finding ways to minimize their environmental footprint as they make life-saving drugs. “In fighting one pathological system, it makes sense to not create another problem that can have a global effect,” Walker said. Plus, Walker said assessing the Taxol pathway enzymes opens doors to new, more natural ways to make Taxol. He said that learning to genetically modify the qualities of the Pacific yew organism to make tomorrow’s versions of Taxol could mean transferring all the genes – the entire pathway – into a bacterium for large-scale production of the new and improved Taxol, without further depleting the yew plant. “Eventually, it will be cool when we’re able potentially to have bacteria make all of the necessary plant enzymes, and we can sit back and watch E. coli make first- and second-generation Taxol molecules,” Walker said.
Acyltransferases: The
primary objective of this project is to employ directed
mutagenesis on a family of Taxus-derived acyltransferases to
identify and characterize molecular determinants within the
active sites that are putatively critical for catalysis and
regio- and substrate specificity. A conserved motif, Cys, His,
and Asp, is postulated to be involved in acyl group transfer to
the acceptor molecule. By site-directed mutagenesis, we are
currently assessing the critical function of the residues in this
triad in relation to acyl/aroyl transfer. Defining the function
of the transferases will also be accomplished through
DNA-shuffling technology, and the resultant hybrid enzymes will
be characterized ultimately by X-ray crystallographic analysis
and molecular modeling.
The research was funded by MSU College of Natural Science. Walker’s laboratory also is funded by the Michigan Agricultural Experiment Station Source / Credit: Michigan State University / Biochemistry & Molecular Biology
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Biosynthesis
of Neoclerodane Diterpenoids. Neoclerodanes are a diverse class
of diterpenoid compounds of which many have demonstrated
bioactivity. Despite the structural variability and differences
in oxygenation and acylation, each is likely derived from a
common kolavenol precursor. Evolutionarily, it is apparent that
each plant making a neoclerodane has recruited a unique series of
enzymes for constructing its target molecule. However, it is
clear that some enzymes in these divergent pathways are similar;
therefore, it is intriguing to assess if, for example, gene
orthologs from Salvia divinorum, that makes κ-opioid
receptor agonist salvinorin A (1), encode protein that catalyze
novel conversion of metabolites occurring in the pathway to
compound 3.
