Blanchard Lab
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:

Lysine Biosynthesis: We succeeded in expressing all eight enzymes of the Mycobacterium tuberculosis L-lysine biosynthetic pathway, and have crystallized and solved the three dimensional structures of five enzymes in the pathway, and have mechanistically characterized seven of the eight enzymes. We have provided both structural and functional information to a number of commercial concerns who are screening for inhibitors of one or more enzymes in this critical pathway in bacteria.

Pantothenate Biosynthesis: This approach has been expanded and refined in the last several years to include enzymes in a number of other biosynthetic pathways, including 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).


The pantothenate biosynthetic pathway in M. tuberculosis

Mycothiol Biosynthesis: Mycobacteria synthesize a structurally unique, low-molecular weight thiol termed mycothiol: N-acetylcysteine in amide linkage to the 2-amino group of the pseudo-disaccharide, D-glucosamine-a-1,1-myo-inositol. We have synthesized a truncated homologue of this compound, and have used it to identify a unique mycobacterial flavoprotein reductase which uses this substrate in oxidative stress management. The mechanistic and structural characterization of the enzymes involved in this biosynthetic pathway is ongoing. The chemistries involved in this pathway bridge the other projects ongoing in the laboratory, including ATP-dependent amide bond formation (see above) and acetyl transfer chemistry (see below).

The mycothiol biosynthetic pathway in M. tuberculosis

 

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 below). 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.

Regioselective acetylation
of 6'-amino group of Tobramycin
 

Protein N-acetyltransferases.

These 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 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 also 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.

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.