Dr. Cecilia A. McIntosh
Professor, Biological Sciences

Dean, School of Graduate Studies

 

mcintosc@mail.etsu.edu

TEACHES:

Teaching Pedagogy for the Graduate Assistant, Mentoring Student Research, Research in Biology

CURRENT RESEARCH PROGRAMS:

The general research areas of my laboratory include plant biochemistry and plant physiology with an emphasis on enzymology, molecular biology, and protein structure/function. My main research interest is in plant natural products and the regulation of their biosynthesis. This research has been supported in part by research grants from the U.S. Department of Agriculture and the National Science Foundation, including a current grant from 2011-15.  The focus of the current grant is to determine the key amino acid residues imparting the strict substrate and regiospecificity of a flavonol-specific 3-O-glucosyltransferase from Citrus paradisi (grapefruit). An overview and summary is presented below.  There have been over 60 undergraduate students, graduate students, and postdoctoral researchers involved in the research program over the last 20 years. Undergraduate and graduate students interested in a research experience are highly encouraged to contact me!

 

Function and Structural Characterization of Putative Secondary Product  Glucosyltransferase Clones from Citrus paradisi
OVERVIEW

The overall research focus of this laboratory has been to study regulation of biosynthesis of specific flavonoids (a major group of secondary metabolites) in order to elucidate factors that control flavonoid synthesis, metabolism, and accumulation during plant development and growth.  One specific focus is elucidation of the regulation of glucosylation of different subclasses of flavonoids resulting in production of the derivatives (e.g. glycosides) actually found in plant tissues. Prior research has supported the hypothesis that biochemical properties of glucosyltransferase (GT) enzymes are a key factor in this process.  There are hundreds of putative secondary product GT genes identified from various plant genome projects; only a small subset of recombinantly expressed enzymes have been biochemically characterized.   

Flavonoids are a group of chemicals made by plants that are involved in flower color, fruit color, and some fruit flavors. Flavonoids sometimes cause insects to avoid eating plants, and some play important roles in attracting insect pollinators. Since plants are such a large part of the human diet, there has also been a lot of research into effects of flavonoids on human physiology. There are nine general types of flavonoids that are made using a common "core" biosynthetic pathway. Modifying reactions can also occur giving each compound specific chemical characters. Due to the variety that can be introduced by modifying reactions, over 6000 different flavonoids are found in plants. No single plant makes all of these; rather there is a specific pattern to the flavonoids made by any one plant type. Plants that can make a variety of flavonoids actually may accumulate one particular compound. For example, 70% of the dry weight of young grapefruit leaves and fruits comes from one single chemical - the bitter flavanone naringin. To make the bitter compound naringin, there is a step-wise addition of two sugars to a compound called naringenin.  The first sugar added is a glucose and this reaction is catalyzed by a flavanone-specific 7-O-glucosyltransferase. In the next reaction, a rhamnosyltransferase adds a rhamnose sugar to a specific position on the glucose.  So, adding two sugars actually makes the compound bitter! Grapefruit are also known to make other flavonoid glycosides and enzymes catalyzing these other reactions have been found in grapefruit leaves and other tissues.  This makes grapefruit a nice model plant to study the structure and function of different flavonoid glucosyltransferase enzymes.  Once the structure/function relationships for these enzymes are elucidated, it may be possible to develop plant varieties with enhanced or new abilities to produce flavonoids such as insect feeding deterrents, medicinal compounds, or desirable flavor and color components. It may also be possible to decrease the plant's production of undesirable compounds.

PROJECT SUMMARY

There are reports of GTs that are highly specific for flavonoid class (or even a subset of compounds within a class) and/or regiospecific for point of glucose addition onto the substrate, and there are reports of enzymes for which results indicate a more promiscuous nature.  Identification and characterization of new GTs coupled with structure/function analysis provides additional information to address questions regarding the impact of particular structural features on substrate and regiospecificity.  Results are used to refine models and perhaps provide information for custom design of GT enzymes.  In order to increase the diversity of information available, this program uses Citrus paradisi as the model plant system.  Young grapefruit leaves have been shown to possess different GTs with ability to glucosylate chalcones, flavanones, flavones, flavonols, and dihydroflavonols.  Some of the GTs are highly specific and possess differential response to inhibitors.   We have been working on obtaining clones for grapefruit GTs and determining function and biochemical properties.  Specific goals of this research are to test hypotheses related to regulation of secondary product metabolism by the biochemical characterization and elucidation of biochemical regulation of proteins expressed by the putative secondary product GT clones. We are also addressing questions pertaining to GT structure/function by determining whether or not particular structural feature(s) of the grapefruit proteins, specifically flavonol-specific 3-O-GT, are responsible for their unique properties.  This will make significant contribution to the field of secondary product structure/function in general and to grapefruit biochemistry and metabolism in particular.

The approach employed to further this work was to use different cloning strategies to obtain candidate grapefruit secondary product GT clones for subsequent heterologous expression and biochemical characterization.  We obtained 11 putative GT clones, one of which coded for a flavonol-specific 3-O-GT, one coded for a simple phenolic GT with broad specificity, and another coded for a GT with limited activity with flavonoid substrates.

We are focusing our structure/function studies on the clone encoding for the flavonol-specific 3-O-GT.  We have cloned the gene into a yeast expressions vector and are using site-directed mutagenesis to alter amino acids to test hypotheses related to critical amino acid residues imparting substrate and regiospecificity.  To date, we have constructed over 30 mutant clones and are expressing the proteins in yeast, purifying, and performing thorough biochemical characterization.

Selected Lab Photos: Past and Present

2007-2009 Lab Staff

2007-09: composite photo, from left to right: Zhangfan Lin (M.S. student), V. “Siddhu” Mallampalli (M.S. student), Fei Han (undergraduate exchange student), Sean Bowen (Undergraduate ETSU Research Discovery student, 2007-08), Brett Pearson (Undergraduate ETSU Research Discovery student and University Honor’s student), Jala Daniel (M.S. student, currently faculty member at Roan State Community College), Mat Halter (NSF REU student), Dr. McIntosh, Patricia Campbell (NSF REU student, 2007-08), Dr. Daniel Owens (NSF Postdoctoral Research Associate), Josephat Asiago (NSF REU student, 2007-08, currently in Ph.D. program at Purdue).

2013-14: Dr. Shivakumar Devaiah (Postdoctoral Research Associate), Ms. Lisa Carter (NSF summer REU student), Mr. Olusegun A. Adepoju (M.S. Student, currently in Ph.D. program at Va. Tech), Ms. Preethi Sathanangam (M.S. Student), group photo including Cheng Zhang (UG student, currently in graduate program in Chicago), Bruce Williams (NSF REU student, currently in ETSU Gatton College of Pharmacy), and Peri Loftis (NSF REU student, currently in ETSU Quillen College of Medicine).

 

SELECTED PUBLICATIONS

BOOK CHAPTERS/INVITED REVIEWS:

Mansell, R.L. and C.A. McIntosh. (1991) 'Citrus spp.: In Vitro Culture and the Production of Naringin and Limonin', in Biotechnology of Medicinal and Aromatic Plants, vol 3, ed. by Y.P.S. Bajaj, Springer-Verlag Berlin, Heidelberg, New York pp 193-210.

Oliver, D.J. and C.A. McIntosh. (1995) 'The Biochemistry of the Mitochondrial Matrix.' in The Molecular Biology of Plant Mitochondria, ed. by S. Levings and I. Vasil, Kluwer Academic Publishers, The Netherlands. pp. 237-280.

McIntosh, C.A. (2000) 'Quantification of Limonin and Limonoate A-ring Monolactone During Growth and Development of Citrus Fruit and Vegetative Tissue by Radioimmunoassay', Chapter 6 in Citrus Limonoids: Functional chemicals in agriculture, food, flavor, and health, ed. by M. Berhow, S. Hasegawa, and G. Manners, ACS Symposium Series.  pp. 73-95.

McIntosh, Cecilia A. (2006) “Translational Opportunities in Plant Biochemistry”, in Integrative Plant Biochemistry as We Approach 2010, Recent Advances in Phytochemistry, vol. 40, J.T. Romeo (ed.), Elsevier Press, New York. pp 307-318.

Owens, Daniel K. and Cecilia A. McIntosh (2011) “Biosynthesis and Function of Citrus Glycosylated Flavonoids”, Recent Advances in Phytochemistry, vol. 41:67-95 (Invited, Peer-reviewed).

 

 

ARTICLES (selected and related to this topic; student authors are underlined)

McIntosh, C.A. and R.L. Mansell. 1990. Biosynthesis of naringin in Citrus paradisi: UDP-glucosyltransferase activity in grapefruit seedlings. Phytochemistry29:1533-1538.

McIntosh, C.A., L. Latchinian, and R.L. Mansell. 1990. Flavanone-specific O-7 glucosyltransferase activity in Citrus paradisi seedlings: Purification and characterization. Arch. Biochem. Biophys. 282:50-57.

McIntosh, C.A., and R.L. Mansell. 1997.  Three-Dimensional Analysis of Limonin, Limonoate A-ring Monolactone, and Naringin in the Fruit of Three Varieties of Citrus paradisi. J. Agric. Food Chem. 45:2876-2883.

Durren, R.L. and C.A. McIntosh. 1999. Flavanone-7-O-Glucosyltransferase Activity from Petunia hybrida. Phytochemistry 52:793-798.

Owens, D.K., T. Hale, L.J. Wilson, and C.A. McIntosh.  2002. Quantification of Dihydrokaempferol Production by Flavanone-3-Hydroxylase using Capillary Electrophoresis.  Phytochemical Analysis 13:69-74.

Pelt, J., W.A. Downes, R. Schoborg, and C.A. McIntosh.  2003. Flavanone 3-Hydroxylase (F3H) Expression in Citrus paradisi and Petunia hybrida Seedlings. Phytochemistry  64:435-444.

RoySarkar, T., C.L. Strong, M.B. Sibhatu, L.M. Pike, and C.A. McIntosh.  2007. Cloning, Expression, and Characterization of a Putative Flavonoid Glucosyltransferase from Grapefruit (Citrus paradisi) Leaves. In Proceedings of the 3rd International Congress on Plant Metabolomics, Iowa State University, B. Nikolau (ed.), Springer Publishing, The Netherlands.  pp. 247-259 (Invited submission)

Knisley, D., E. Seier, D. Lamb. D.K. Owens, and C.A. McIntosh. 2009.  A Graph-Theoretic Model Based on Primary and Predicted Secondary Structures Reveals Functional Specificity in a Set of Plant Secondary Product UDP-Glucosyltransferases.   Proceedings of the 2009 International Conference on Bioinformatics, Computational Biology, Genomics, and Chemoinformatics. W. Logong, M. Doble, Z. Sun, and J. Malone(eds.), pp 65-72 (Peer-reviewed, Invited).

Owens, D.K. and C.A. McIntosh.  2009. Identification, Recombinant Expression, and Biochemical Characterization of a Flavonol 3-O-Glucosyltransferase Clone from Citrus paradisi.  Phytochemistry 70:1382-1391.

Jackson, A.R., D. Knisley, C. McIntosh, and P. Pfeiffer. 2011. Predicting Flavonoid UGT Regioselectivity.  Advances in Bioinformatics  2011( ID 506583): 15 pp.  (doi:10.1155/2011/506583)

 Daniel, J.J., D.K. Owens, and C.A. McIntosh.  2011. Secondary Product Glucosyltransferase Expression During Citrus paradisi Growth and Development. Phytochemistry 72:1732-1738. 

McIntosh, C.A., K. Bartoszuk, and B.J. Maxson.  2014 Integration and Synergy of Research and Graduate Education in Science, Humanities, and Social Sciences. Critical Conversations: An Interdisciplinary Journal 1:70-92. (invited, peer-reviewed).

Devaiah, S.K., D.K. Owens, M.B. Sibhatu, T. Roy Sarkar, C.L. Strong, V.K.P.S.Mallampalli, J. Asiago, J. Cooke, S. Kiser, Z. Lin, A. Wamucho, D. Hayford, B.E. Williams, P. Loftis, M. Berhow, L.M. Pike. and C.A. McIntosh.  2015.  Isolation, Recombinant Expression and Biochemical Characterization of Putative Secondary Product Glucosyltransferase Clones from Citrus paradisi.  IN PRESS

Devaiah, S.K., B.M. Tolliver, and C.A. McIntosh. Structural and functional study of flavonol-specific glucosyltransferase from Citrus paradisi using site-directed mutagenesis. In Preparation.

 

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This website was designed by Cecilia McIntosh
Last Update: January 2015