D1. GDP-mannose 4,6-dehydratase (GMD) assay development and pH rate profiles
This project will enable students to function as competent enzymologists through the
development of a novel assay utilized in the measurement of the kinetics of GMD while
developing a comprehensive appreciation of the intricacies of glycobiology and an
understanding of translational research as a postdoctoral option.
Primary Faculty co-Advisors
Grover L. Waldrop, Ph.D., Biochemistry (Enzymology)
Roger A. Laine, Ph.D., Biochemistry (Carbohydrate Chemistry)
Off-campus Participant: Ralph Freedman, M.D., Ph.D., UT-Houston, MD Anderson (Antitumor immunity in humans)
Technical Proposal: In both prokaryotes and eukaryotes the primary biosynthetic route to
GDP-fucose is from GDP-mannose. This transformation is accomplished by two
enzymes, GDP-mannose 4,6 dehydratase (GMD) and GDP-fucose synthetase (GFS).
The focus of this study is GDP-mannose 4,6-dehydratase (GMD), which catalyzes the
reaction shown in Figure 1.
Figure 1
The initial step of this reaction involves the oxidation of the C4 position of the mannose
ring to a keto-functionality followed by the reduction of the C6 carbon to a methyl group.
This concerted oxidation and reduction involves an intramolecular hydride transfer from
the C4 to the C6 on the mannose ring by a tightly bound NADP cofactor, which becomes
transiently reduced during the course of the reaction.
The reaction catalyzed by GMD is a novel target for inhibition and could generate a nonsteroidal anti-inflammatory and/ or anti-metastatic pharmaceutical. After injury and
before bacterial infection, there are several processes required in initiating the immune
response with the principal event being selectin mediated cellular adhesion. Injured cells
adjacent to endothelial cells launch distress signals (chemoattractants); endothelial cells
are triggered (in seconds) to exocytose P-selectin. Leukocytes constitutively display
selectin ligands (or selectin counter receptors) see Figure 2.
Figure 2
Selectins on endothelial cells bind selectin ligands on leukocytes making cellular
attachment possible. Selectin binding selectin ligand is mediated by the carbohydrate
recognition domain on selectin interacting with the fucose on the selectin ligand (Figure
3). There are three types of selectins E, endothelial, P, platelets, and L- leukocyte, each a
membrane spanning receptor protein.
Figure 3
In the absence of fucosylated selectin ligands endothelial cells do not adhere to
leukocytes; if there is no adhesion between endothelial cells and leukocytes then there is
no inflammatory response. Thus inhibition of any enzyme involved in the synthesis of
fucose is going to diminish the inflammatory response.
It is now clear that adhesive interactions play a critical role in the process of metastatic
tumor dissemination. Adhesion molecules act as both positive and negative modulators of
the metastatic process. Because tumor cells are rapidly eliminated from the circulation,
those cells that can quickly arrest in the vasculature at a secondary site and pass through
the vessel wall into the surrounding tissue will have a selective advantage toward
establishing new metastatic colonies. The first step in this process is specific adhesion to
venular endothelial cells in selected organs, a process mediated by tumor cell surface
molecules such as the selectin ligand carbohydrate domain mediate binding to endothelial
adhesion molecules such as the E-selectin. (5) Thus inhibiting fucose synthesis also has
implications affecting the metastatic process.
Prior to designing a pharmaceutical inhibitor that targets GMD, the basic chemistry of
this catalysis needs to be understood. This project has three specific aims: (1) Develop a
facile fluorescent assay for GMD, (2) Test the roles of residues Tyr 157 and Glu135 in
acid base catalysis by GMD, (3) Screen 80 GDP-D-mannose structural analogs for
inhibition of GMD. A brief discussion of each specific aim follows.
1) At this time there is no facile assay for the conversion of GDP-D-mannose to GDP-4keto, 6-deoxy-D-mannose. Our first objective is to develop a precise, convenient, and
rapid assay for the reaction catalyzed by GMD. We will assay GMD by measuring
formation of the product GDP-4-keto, 6-deoxy-D-mannose using a fluorescent dye that
binds ketones called 5-dimethylaminonaphthalene- 1-sulfonyl hydrazine also known as
dansyl hydrazine (3). The fluorescence of dansyl hydrazine will be measured with a
Spectra Max Gemini Fluorescent Plate Reader by Molecular Devices.
2) The assay developed in specific aim one will be used to test the roles of residues Tyr
157 and Glu135 in acid-base catalysis by GMD (2). The proposed mechanism of GMD
(Figure 4) involves the initial abstraction of a hydride by NADP from the C4 position of
the mannose ring. During this step Tyr157 plays the role of active-site base deprotonating
the 4-hydroxyl group. Next, Glu135 functions as a general base to remove the C5 proton
during the dehydration step leading to formation of the 5,6 mannoseen intermediate.
It is hypothesized that Tyr 157 acts as a catalytic base, while Glu135 acts as a catalytic
acid; we will test this hypothesis by substituting a Val for Tyr157 and Gln instead of
Glu135. The pH rate profile of both mutant enzymes will be determined and compared to
the native enzyme.
Figure 4
The kinetic parameters V/max and Vmax/ Km will be determined at pH intervals of 0.4.
We anticipate there will be a half-bell curve on the acid side with a slope of 2.
3) Finally, we will test 80 compounds that are structural analogs of GDP-mannose as
potential inhibitors of GMD. We will determine the binding constant (Ki) and the type of
inhibition: competitive, noncompetitive, uncompetitive with respect to GDP-mannose for
the compound that best inhibits GMD.
Two IGERT apprentices to be recruited: one maintaining a Biochemistry
prospectus and a second within the Chemistry department.
This venture is consistency with the Macromolecular Education, Research & Training
theme because it incorporates two diverse faculties Dr. Waldrop, an enzymologist and Dr.
Laine, a glycobiologist, who can guide a project centered around an enzyme catalyzing the
conversion of a carbohydrate.
How does the project form a vector cross-product of existing research themes by the
participants?
Existing research directions. Dr. Waldrop research focuses on the kinetic and chemical
mechanisms of enzymes. Currently, the laboratory is focusing on the catalytic mechanism of
acetyl CoA carboxylase. Acetyl CoA carboxylase catalyzes the committed and regulated step in
fatty acid synthesis; the first reaction involves the carboxylation of the vitamin biotin followed
by the transfer of the carboxyl group from biotin to acetyl CoA to form malonlyl CoA. To study
the catalytic mechanism he is using a variety of mechanistic techniques including steady-state
kinetics, inhibitor design, isotope effects and site-directed mutagenesis. Dr. Laine is interested
in Bioactivity and structure of complex carbohydrates, including structural characterization
using mass spectrometry and other methods. Furthermore, cell surface carbohydrates of
microbes and eukaryotes are studied with a focus on use of these saccharides for detection by
analyte specific probes such as antibodies, lectins and enzymes. His is an entrepreneur and
keenly interested in microbial and fungal diagnostics using catalytically disabled
endoglycosidase binding assays and is intrigued by the biochemistry of the inflammatory
process. He has also directed a research project, led by Dr. Narasinga Rao in Glycomed, Inc., in
the 1990’s that helped discern the pharmacophore of the selectins. Dr. Freedman takes a
"translational" approach in that he seeks to transfer new knowledge to patients through
innovative clinical trials, while taking the opportunity to participate in the discovery process.
His basic science program is studying factors in the tumor microenvironment that can modify
the immune response in vivo.
New research direction. It is unlikely that these professors would otherwise work together,
considering there was no collaboration in the past nor is the idea of collaboration apparent. The
possibility of this team coming together other than described and working on this project is
nonexistent. Dr. Waldrop will now assist the team in creating an assay for an enzyme that
catalysis a reaction entirely unrelated to his traditional work. Dr. Laine will investigate
detection methods (dyes) for the substrate and product. Dr. Freedman will serve as an advocate
for potentially viable augmentations relating this project to human pathology.
How do students benefit from the team-oriented research, beyond what would be available
to them from either advisor separately? See examples in Appendix.
Briefly describe the support level available to each individual faculty or off-campus
participant (i.e., without IGERT). Dr. Laine has $265,000/year shared with Prof. Gregg
Henderson, from the USDA and state sources, mainly for termite-related research. Some may
appreciate the apposite belief that, “Silver and Gold Have I None; but Such as I Have Give I
Thee”, the fruits of this work will be beholden to IGERT.
Interdisciplinary strengths of the team project: This project is an exceptional collaboration of
multiple disciplines; it is borne of basic research while paving a potential route for translational
application towards a biomedically relevant conclusion, namely the potential for an antiinflammatory/ anti-metastatic pharmaceutical. This project exemplifies characterization of
macromolecules which is crucial to understand human diseases. As projected, kinetic
characterization of GDP-mannose 4,6-dehydratase via pH rate profiles and inhibition screening,
utilize expertise of three distinguished researchers, an enzymologist, glycobiologist, and a
physician-scientist.
Commitment of faculty & off-campus participants to work side-by-side with apprentices:
Each professor plans to commit substantial resources to the student, especially time spent in
teaching techniques, at the laboratory bench and in education pertaining to theoretical
underpinnings during the apprenticeship period. Professor Waldrop, an associate professor
within the Department of Biology, promises an average of 10 hours weekly to demonstrate
techniques and monitor the project progress. Professor Laine, a full professor in the
Department of Biology will be available for questions related to the usefulness and technical
guidance of the equipment at his disposable, i.e. flowcytometer/ plate reader, HPLC.
Dr.Freedman provides unique opportunities for students, both in the basic science and clinical
programs, to participate and to contribute in these new and exciting discoveries.
References:
1.
2.
3.
4.
5.
6.
Sullivan, F, Kumar, R, Kriz, R, Stahl, M, Xu, G, Rouse, J, Chang, X, Boodhoo, A, Potvin, B,
Cumming, D: “Molecular Cloning of Human GDP-mannose 4,6-Dehydratase and Reconstitution
of GDP-fucose Biosynthesis in Vitro” Jor Biol Chem, 1998 273/ 14, 8193-8202
Somoza, J, Menon, S, Schmidt, H, Joseph-McCarthy, D, Dessen, A, Stahl, M, Somers, W and
Sullivan, F: “Structural and kinetic analysis of Escherichia coli GDP mannose 4,6 dehydratase
provides insights into the enzyme’s catalytic mechanism and regulation by GDP-fucose” Structure,
2000, 8, 123-135
GIVE REFERANCE OF DYE’S UTILITY
Nakayama, K, Maeda1, Y, Jigami, Y: “Interaction of GDP-4-keto-6-deoxymannose-3,5-epimerase
reductase with GDP-mannose-4,6-dehydratase stabilizes the enzyme activity for formation of
GDP-fucose from GDP-mannose” Glycobiology, 2003, 13/ 10, 673-680 (NEED TO JOIN TO
APPLICABLE INFO)
Zetter BR., “Adhesion molecules in tumor metastasis”, Seminars in Cancer Biology 1993
Aug;4(4) 215-229
Dr. Waldrop and Dr. Laine in critique