Professor, Department of Biochemistry
Dan Danciger Professor of Biochemistry
John S. Blanchard Research Interests The treatment of bacterial infections with antibiotics is being severely compromised by the rapid development and dissemination of drug resistance and has become of significant clinical concern. Through a combination of recombinant DNA methods, protein purification, kinetic and chemical mechanistic analysis and three-dimensional structural description, we are developing several enzymes into validated targets for subsequent inhibitor, and potential drug design. Essential Bacterial Biosynthetic Pathways Bacteria must produce all twenty amino acids and all organic enzyme cofactors that were identified as vitamins. Many of these pathways are known to be essential for bacterial survival, and the pantothenate pathway is one such pathway. Insertional inactivation of the panC&D genes results in strains of M. tuberculosis that are highly attenuated.
We have focused on the enzymological characterization of the early steps, unique to bacteria and plants, of Coenzyme A biosynthesis, an essential pathway in M. tuberculosis. Starting with the condensation of two molecules of pyruvate, pantothenate is generated by a series of enzymatic steps with few precedents in biology. These include one of two examples of a B12-independent 1,2-methyl shift (acetolactate isomeroreductase), a reductive formyltransfer (ketopantoate hydroxymethyltransferase) and one of only a few examples of an ATP-dependent amide forming reaction (pantothenate synthetase). Our enzymatic studies have been spurred by the recent determination of the three-dimensional structures of most of the enzymes in this pathway. Antibiotic Drug Resistance Studies from this laboratory were responsible for the identification of the target of the most widely used anti-tubercular drug, isoniazid, as being the M. tuberculosis enoyl-ACP reductase.
More recently, our efforts have turned to the determination of the mechanisms of resistance to broad-spectrum antibiotics, in particular the aminoglycoside class of antibacterials. Clinical resistance to aminoglycosides is due to the expression of genes encoding enzymes that chemically modify the drug, either by phosphorylation, adenylylation or acetylation. By far the most prevalent of these mechanism is N-acetylation, catalyzed by a wide range of enzymes that exhibit regioselective acetylation. We are actively studying both the M. tuberculosis 2’-acetyl-transferase, that can uniquely catalyze both 2’-O- and N-acetylation, and the S. enterica 6’-acetyltransferase (regioselectivity of the reaction shown at right).
The structures of these enzymes in complex with CoA and ribostamycin has allowed us to define the molecular basis for the observed regioselectivity. Both of these enzymes are chromosomally-encoded, suggesting that they have evolved their antibiotic-modifying activity from an extant, but unknown, activity. Protein N-acetyltransferases The aforementioned studies have led us to an examination of the GNAT superfamily of N-acetyltransferases, of which both the prokaryotic aminoglycoside N-acetyltransferases and eukaryotic histone acetyltransferases are members. Of the twenty-six GNAT superfamily members in E. coli, only one has been expressed and functionally characterized, three are ribosomal protein N-acetyltransferases, and the other 22 have unknown function. These small (120-200 aa) enzymes can be identified from four sequence motifs that represent the core “fold” of the N-acetyltransferases, of which some 10,000 superfamily members can be identified in sequenced genomes. The known monomer fold structures are nearly identical, and represent an acetyl-CoA binding domain.
The specificity for the acetylatable substrate is determined both by residues of the monomer, but also by residues at the dimer interface, which are vastly different for different GNAT’s. The determination of the three-dimensional structures of these enzymes, and their ability to N-acetylate both small molecules, peptides and proteins suggests that these enzymes have have multiple substrates and physiological functions. In order to determine the physiological substrates for the genomic ensemble of GNAT’s, we have developed a method and reagent which has allowed us to covalently modify the substrate of any acetyltransferase. The reagent, chloroacetyl-CoA (ClAcCoA) is a substrate for the acetyltransferase, and the chloroacetyl group is transferred to the substrate to generate the chloroacetylated product. The other product, CoASH, is a potent nucleophile, which attacks the a-chloroacetylated product to generate an acetyl-CoAylated substrate. When [3’-32P]-ClAcCoA is used, the product of the reaction becomes radiolabeled, and for protein substrates, can be observed after SDS-PAGE and autoradiography. We have shown that this reagent can be used to label ribosomal protein L12 in the presence of the rimL-encoded N-acetyltransferase.
We have also demonstrated that exogenously added thiol-containing compounds containing both fluorecent probes and affinity labels can be used to identify both the known protein substrates of N-acetyltransferases and other unknown substrates in cell extracts. We wish to expand these studies to include the identification of all the substrates for all the N-acetyltransferases in both Salmonella enterica and Mycobacterium tuberculosis. We have also recently demonstrated that the yeast Hat1 histone acetyltransferase will use ClAcCoA as a substrate, and will selectively chloroacetylate histones H3 and H4 in a mixture of yeast histones. We now believe that the reagent will be useful in defining the expanding manifold of eukaryotic transcription factors whose acetylation status, like those of the histones themselves, regulates transcription and the activity and stability of the eukaryotic transcription apparatus.
Finally, we are developing new methods to identify the large number of proteins that are known to be post-translationally modified by lipidation, including both myristoylation and palmitoylation. Suitability for Undergraduate, Graduate and Postdoctoral Trainees The projects described above are eminently suitable for all levels of scientific trainees. Although the laboratory today has a preponderance of postdoctoral fellows, and a single graduate student, summer undergraduate students have participated in the research program and been co-authors in a number of published works. Undergraduate students with an interest in biological chemistry, and a strong background in organic and physical chemistry, would be especially qualified to participate in the research program.
Mechanistic and Kinetic Study of the ATP-dependent DNA Ligase of Neisseria meningitidis. Magnet, S. and Blanchard, J. S. (2004) Biochemistry 43, 710-717.
Aminoglycoside Microarrays to Study Antibiotic Resistance. Disney, M. D., Magnet, S., Blanchard, J. S. and Seeberger, P. H. (2004) Angew. Chemie Int’l Ed. 43, 1591-1594.
A Bacterial Acetyltransferase Capable of Regioselective N-Acetylation of Antibiotics and Histones. Vetting, M.W., Magnet, S., Nieves, E., Roderick, S.L and Blanchard, J.S. (2004) Chemistry & Biology 11, 565-573 [cover illustration].
Kinetic and Chemical Mechanism of Mycobacterium tuberculosis 1-Deoxy-D-xylulose-5-phosphate Isomeroreductase. Argyrou, A. and Blanchard, J. S. (2004) Biochemistry 43, 4377-4384.
Active Site Residues in Mycobacterium tuberculosis Pantothenate Synthetase Required in the Formation and Stabilization of the Adenylate Intermediate. Zheng, R., Dam, T., Brewer, C.F. and Blanchard, J.S. (2004) Biochemistry 43, 7171-7178.
Characterization of a new Member of the Flavoprotein Disulfide Reductase family of Enzymes from Mycobacterium tuberculosis. Argyrou, A., Vetting, M.W. and Blanchard, J.S. (2004) J. Biol. Chem. 279, 52694 - 52702.
Amoxicillin-Clavulanate Therapy Increases Childhood Nasal Colonization by Methicillin-Susceptible Staphylococcus aureus Strains Producing High Levels of Penicillinase. Guillemot, D., Bonacorsi, S., Blanchard, J.S., Weber, P., Simon, S., Gueson, B., Bingen, E. and Carbon, C. (2004) Antimicrob. Agents Chemother. 48, 4618-4623.
General Metabolism and Biochemical Pathways of Tubercle Bacilli. Wheeler, P. W. and Blanchard, J. S. in “Mycobacterium tuberculosis” (S.T. Cole, W.R. Jacobs, Jr, and B. Gicquel, eds.) ASM Press, 2004.
Flavoprotein Disulfide Reductases: Advances in Chemistry and Structure. Argyrou, A. and Blanchard, J. S. (2004) Progress in Nucleic Acid Research and Molecular Biology, Vol. 78, 89-142.
Structure and Functions of the GNAT Superfamily of Acetyltransferases. Vetting, M.W., de Carvalho, L.P., Yu, M., Hegde, S.S., Magnet, S., Roderick, S.L. and Blanchard, J.S. (2005) Arch. Biochem. Biophys. 433, 212-226.
Molecular Insights into Aminoglycoside Mechanism and Resistance. Magnet, S. and Blanchard, J.S. (2005) Chem. Rev., in press.
More Information About Dr. John Blanchard
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Albert Einstein College of Medicine
Jack and Pearl Resnick Campus
1300 Morris Park Avenue
Forchheimer Building, Room 315
Bronx, NY 10461
The Wall Street Journal features comments from Dr. John Blanchard on two possible Nobel Prize winners for this year.
Associated Press, U.S. News & World Report, Reuters (India and UK), Scientific American, BBC and others interview Dr. John Blanchard regarding his groundbreaking tuberculosis study published in the journal Science.