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Michael G. Malkowski, Ph.D EDUCATION |
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ADDRESS: Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203-1102 |
CONTACT
INFORMATION: Tel. 716-898-8624 Fax. 716-898-8660 E-mail: malkowski@hwi.buffalo.edu |
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Research Interests Crystallographic characterization and functional analysis of integral membrane enzymes involved in lipid metabolism; enzymatic mechanisms of fatty acid oxygenation; development of tools for high-throughput structural characterization of membrane proteins The Structural Biology of Oxylipin Biosynthesis Public perception of healthy food and lifestyle, as well as the identification of specific biological roles in certain clinical conditions, has brought a renewed attention to polyunsaturated fatty acids (PUFAs) with respect to health and disease. PUFAs are lipid molecules that differ in hydrocarbon length and degree of saturation. These essential fatty acids cannot be synthesized de novo by most eukaryotic organisms and therefore must be obtained from dietary sources. Once ingested, PUFAs are acted upon by desaturase and elongase enzymes, which form a larger family of precursors that play a key role in maintaining the fluidity and structural integrity of all cell membranes. PUFAs play an equally significant role as signaling molecules in numerous biologically vital pathways. However, in order to exert their full range of biological effects, PUFAs are generally metabolized into more potent substances called oxylipins. The term “oxylipin” is used collectively to refer to oxygenated, 18-22 carbon PUFA-based compounds that are biosynthesized through the stereospecific addition of one, two, or four atoms of oxygen at different carbon positions, followed by subsequent conversion of the developing peroxide or hydroperoxide intermediate into a stable, biologically active compound. Arachidonic acid (AA; 20:4 w-6) is the most vital PUFA precursor of the eicosanoid class of oxylipins in animals. The “arachidonate cascade”, which is comprised of the cyclooxygenase, lipoxygenase, and epoxygenase pathways, is responsible for the production of prostaglandins, leukotrienes, lipoxins, hydroxy-, and epoxy- eicosatetraenoic acids. These potent lipid signaling molecules are synthesized and released rapidly in response to extracellular stimuli, where they play an important role in a number of physiological processes. Moreover, they play a major role in a vast array of inflammatory disorders such as asthma, rheumatoid arthritis, atherosclerosis, and can be linked to other diseases that are associated with inflammation including cardiovascular disease, Alzheimer's disease, and cancer. The major focus of my research is to use biochemical analyses, biophysical tools, and x-ray crystallographic methods to understand how different classes of integral membrane enzymes carry out stereoselective oxygenations to generate unique oxylipins from a defined set of PUFA substrates. To gain insight on the chemical determinants that drive stereoselective oxygenation of PUFAs at the molecular level, we are studying different integral membrane enzymes involved in oxylipin production in plants, animals, and invertebrates.
Characterizing the Structural Determinants Involved in the Stereoselective Oxygenation of 18-Carbon PUFAs by Pathogen Inducible Oxygenase (PIOX), a Plant Fatty Acid Alpha Dioxygenase
Understanding the Stereoselective Oxygenations of AA by Aspirin-Acetylated COX-2 Our goal is to understand the molecular mechanism by which Cyclooxygenase-2 (COX-2) stereospecifically oxygenates AA to form "novel" (15R)-prostaglandins (15R-PGs). COX-1 and COX-2 catalyze the committed step in PG biosynthesis and are the pharmacological targets of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), which provide relief from the inflammation, swelling, and pain associated with arthritis. In contrast to all other NSAIDs, aspirin covalently modifies COX-1 and COX-2 via the acetylation of a critical serine residue within the cyclooxygenase active site, resulting in complete inhibition of PG biosynthesis. However, aspirin acetylation of COX-2 results in a shift in the reaction specificity of the enzyme, converting it from a cyclooxygenase to a lipoxygenase, resulting in the formation of (15R)-hydroxyeicosatetraenoic acid (15R-HETE). 15R-HETE is converted by downstream enzymes into (15R)-epi lipoxin, an aspirin-triggered lipid mediator with potent anti-inflammatory actions. We are using a combination of site-directed mutagenesis, functional analyses, and x-ray crystallographic methods to test the hypothesis that the inversion of stereochemistry at carbon-15 observed in products formed by aspirin acetylated COX-2 is a result of AA binding in an "unconventional" conformation within the cyclooxygenase active site prior to the initiation of catalysis. These studies will provide for a complete mechanistic understanding of how 15R-PGs (and ultimately aspirin-triggered lipoxins) are generated, lend valuable insight into how COX enzymes stereospecifically control oxygenation during catalysis, and lead to further exploration and development of new or combined therapeutic approaches for the treatment of arthritis, with fewer unwanted side effects. The Development of High-Throughput Methods to Crystallize Membrane Proteins Elucidating the structures of membrane proteins at atomic resolution is essential to our understanding of disease states and a critical component in the rational design of novel and more effective drugs. Crystallographic characterization of a membrane protein begins with detergent solubilization of the protein from the lipid bilayer and subsequent purification of a functionally stable, homogeneous protein-detergent complex (PDC). Crystallization of the subsequent PDC requires the manipulation of the dual surface properties of the PDC in the presence of various precipitants. As membrane proteins have been observed to form crystals close to the phase separation boundaries of the detergent, knowledge of these boundaries under different chemical conditions can serve as a foundation for the design of rational crystallization screens. The Hauptman-Woodward Medical Research Institute provides a high-throughput crystallization screening service for both the structural genomics and biological crystallographic communities. An important goal of the HTS laboratory is to develop novel technologies for expression and crystallization of membrane proteins and other difficult to crystallize biological macromolecules within the National Institutes of General Medical Sciences Protein Structure Initiative. We have generated empirically derived detergent phase data for an extensive chemical landscape encompassing 11 detergents, 11 salts, and 10 PEGs. The resulting data were used to formulate a membrane protein crystallization screen. The screen uses 1536 cocktails to sample regions of detergent phase space particularly relevant to crystallization. The membrane protein screen is offered as an alternative to the set of 1536 cocktails in use by the HT screening laboratory for soluble proteins. Selected Publications Pieper U, Schlessinger A, Kloppmann E, Chang GA, Chou JJ, Dumont ME, Fox BG, Fromme P, Hendrickson WA, Malkowski MG, Rees DC, Stokes DL, Stowell MH, Wiener MC, Rost B, Stroud RM, Stevens RC, Sali A. Coordinating the impact of structural genomics on the human α-helical transmembrane proteome. Nat Struct Mol Biol. 2013 Feb;20(2):135-8. PMID: 23381628 Goulah CC, Zhu G, Koszelak-Rosenblum M, Malkowski MG. The crystal structure of α-dioxygenase provides insight into diversity in the cyclooxygenase-peroxidase superfamily. Biochemistry. 2013 Feb 26;52(8):1364-72. Epub 2013 Feb 14. PMID: 23373518 Vecchio AJ, Orlando BJ, Nandagiri R, Malkowski
MG. Investigating
substrate promiscuity in cyclooxygenase-2: The role of ARG-120 and
residues lining the hydrophobic groove. J Biol Chem. 2012 Jul 13;287(29):24619-30.
Epub 2012 May 25. [Pub Med ID: 22637474] Luthra A, Zhu G, Desrosiers DC, Eggers CH, Mulay V, Anand
A, McArthur FA, Romano FB, Caimano MJ, Heuck AP, Malkowski MG, Radolf
JD. The transition from closed to open conformation of Treponema
pallidum outer membrane-associated lipoprotein TP0453 involves
membrane sensing and integration by two amphipathic helices. J Biol
Chem. 2011 Dec 2;286(48):41656-68. Epub 2011 Sep 29. [Pub Med ID:
21965687] Vecchio AJ, Malkowski MG. The structure of NS-398
bound to cyclooxygenase-2. J Struct Biol. 2011 Nov;176(2):254-8. Epub
2011 Aug 6. [Pub Med ID: 21843643] Dong L, Vecchio AJ, Sharma NP, Jurban BJ, Malkowski MG, Smith
WL. Human cyclooxygenase-2 is a sequence homodimer that
functions as a conformational heterodimer. J Biol
Chem. 2011 May 27;286(21):19035-46. Epub 2011 Apr 5. [Pub Med ID:
21467029] Vecchio AJ, Malkowski MG. The
structural basis of endocannabinoid oxygenation by cyclooxygenase-2.
J Biol Chem. 2011 Jun 10;286(23):20736-45. Epub 2011 Apr 13. [Pub
Med ID:21489986] Vecchio AJ, Simmons DM, Malkowski
MG. The
structural basis of fatty acid substrate binding to cyclooxygenase-2. J
Biol Chem. 2010 Jul 16:285(29):22152-63. Epub 2010 May 12. [Epub
ahead of print] [Pub Med ID: 20463020] Calamini B, Ratia K, Malkowski
MG, Cuendet M, Pezzuto JM, Santarsiero BD, Mesecar AD. Pleiotropic mechanisms facilitated
by resveratrol and its metabolites. Biochem J. 2010 Jul 15;429(2):273-82.
[Pub Med ID: 20450491] Koszelak-Rosenblum M, Krol
A, Mozumdar N, Wunsch K, Ferin A, Cook E, Veatch CK, Nagel R, Luft
JR, Detitta GT, Malkowski MG. Determination and application of empirically derived
detergent phase boundaries to effectively crystallize membrane proteins. Protein Sci. 2009
Sep;18(9):1828-39. [Pub Med ID: 19554626] Koszelak-Rosenblum, M., Krol, A.C., Simmons,
D.M., Goulah, C.C., Wroblewski, L., and Malkowski, M.G. (2008)
His-311 and Arg-559 are key residues involved in fatty acid oxygenation
in pathogen-inducible oxygenase. J. Biol. Chem., 283,
24962-24971. Snell, E.H., Luft, J.R., Potter, S.A., Lauricella, A.M., Gulde, S.M., Malkowski, M.G., Koszelak-Rosenblum, M., Said, M.I., Smith, J.L., Veatch, C.K., Collins, R.J., Franks, G., Thayer, M., Cumbaa, C., Jurisica, I., and DeTitta, G.T. (2008) Establishing a training set through the visual analysis of crystallization trials part I: ~150,000 images. Acta Cryst. D64, 1123-1130. http://scripts.iucr.org/cgi-bin/paper?S0907444908028047 Snell, E.H., Lauricella, A.M., Potter, S.A.,
Luft, J.R., Gulde, S.M., Collins, R.J., Franks, G., Malkowski, M.G.,
Cumbaa, C., Jurisica, I., and DeTitta, G.T. (2008)
Establishing a training set through visual analysis of crystallization
trials part II: Crystal
examples. Acta Cryst. D64, 1131-1137. Fox, B.G., Goulding, C., Malkowski, M.G., Stewart, L., and
Deacon, A. (2008) Genes to structure with valuable
materials and many scientific questions in between. Nature
Methods, 5, 129-132. Malkowski, M.G., Quartley, E., Friedman, A.E., Babulski, J., Kon, Y., Wolfley, J.R., Said, M.I., Luft, J.R., Phizicky, E.M., DeTitta, G.T., and Grayhack, E.J. (2007) Blocking S-adenosylmethionine Synthesis in Yeast Allows Selenomethionine Incorporation and Multiwavelength Anomalous Dispersion Phasing. Proc. Natl. Acad. Sci., USA. 104, 6678-6683. http://www.pnas.org/content/104/16/6678.long Luft, J.R., Wolfley, J.R., Said, M.I., Nagel, R.M., Lauricella,
A.M., Smith, J.L., Thayer, M.H., Veatch, C.K., Snell, E.H., Malkowski,
M.G., and DeTitta, G.T. (2007) Efficient Optimization
of Crystallization Conditions by Manipulation of Drop Volume Ratios
and Temperature. Protein Science. 16,
715-722. Lloyd, T., Krol, A., Campanaro, D., and Malkowski, M.G. (2006)
Purification, Crystallization, and Preliminary X-Ray Diffraction Analysis
of Pathogen-Inducible Oxygenase (PIOX) From Oryza sativa. Acta
Cryst. F62, 365-367. Garavito, R.M., Malkowski, M.G., and DeWitt,
D.L. (2002) The Structures of Prostaglandin Endoperoxide H Synthases-1 |
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| 700 Ellicott Street Buffalo, New York 14203-1102 Tel: 716 898 8600 Fax: 716 898 8660 | ||||||
Pathogen-Induced Oxygenase (PIOX) is a heme-containing,
membrane-associated protein found in mono- and dicotyledon plats that
utilizes molecular oxygen to convert the PUFAs linoleic acid and alpha
linolenic acid into their corresponding 2R-hydroperoxides. PIOX,
which is a key component of the host-defense response in plants, is
a member of a larger family of fatty acid alpha dioxygenases that includes
mammalian COX-1 and COX-2. Many residues crucial for activity
in mammalian COX are conserved in PIOX. These residues include
the distal and proximal histidine residues involved in heme binding,
and a catalytic tyrosine, which is responsible for the initiation of
catalysis. While there are global differences between PIOX and
COX enzymes with respect to their potential overall structural architecture,
substrate specificities, and signaling pathways, the mechanism by which
these enzymes stereoselectively oxygenate PUFAs are strikingly similar. Our
goal is to elucidate the structure of PIOX and determine the mechanism
controlling catalysis. This information will enhance the existing
knowledge of how PUFAs are stereoselectively oxygenated and provide
useful insights into other systems, which discriminate between the
saturation and chain length of a PUFA substrate