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Research Interests
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| 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. |
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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).
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The pantothenate biosynthetic pathway in M.
tuberculosis
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| 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
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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.
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Regioselective acetylation
of 6'-amino group of Tobramycin |
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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.
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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.
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