In the area of microbial bioterrorism, we (P. Reddy, S-K, Kim and S. Patterson) are working on the characterization of virulence factors to understand the molecular basis of pathogenesis in Yersinia pestis (plague causing bacteria) and Mycobacterium tuberculosis (tuberculosis disease).  Adenylyl cyclase, one of the virulence factors in Bacillus anthracis (anthrax disease), is being used as a model system to understand its role in other bacterial pathogens.

Adenylyl cyclases are classified into three categories. Class I adenylyl cyclases are from enteric bacteria such as Escherichia coli and Salmonella typhimurium. These prokaryotic Class I enzymes are primarily regulated by sugar sensor proteins. Adenylyl cyclase from Yersinia pestis, the plague causing bacteria and a potential bioterrorism agent, has overwhelming homology (85%) with the E. coli enzyme. Class II adenylyl cyclases are from Bacillus anthracis and Bordetella pertussis. Class II adenylyl cyclases are virulent factors in these two bacterial species. Class II enzymes elicit their virulence by producing a supraphysiological concentration of cyclic adenosine monophosphate as a consequence of the activation by the host calmodulin. Class III adenylyl cyclases are highly complex macromolecules containing two spans of six helical transmembrane domains and two cytoplasmic domains. Class II enzymes are regulated by a variety of small molecules such as hormones, guanosine triphosphate, and macromolecular proteins such as guanine nucleotide binding proteins, and calmodulin. Class III enzymes were originally thought to be only from higher eukaryotic origin. But the work from our laboratory has shown that Mycobacterium tuberculosis contains only Class III adenylyl cyclases, albeit half the molecular secondary structure with only one six helical transmembrane domain and one cytoplasmic domain. However the mycobacterial enzyme functions as a dimer mimicking the eukaryotic enzyme. Even more intriguing is the fact that there are 15 genes for adenylyl cyclase spread around the genome of Mycobacterium tuberculosis. We are performing the gene expression and activity profile measurements for these 15 adenylyl cyclase genes.

We (S-K, Kim, S. Patterson, and P. Reddy) have cloned the gene for adenylyl cyclase (850 amino acids) from Yersinia pestis. The catalytic function of this enzyme resides in the amino terminal half of the protein (aa1-450). The catalytic domain was expressed with a histidine tag and purified in a single step. We are presently conducting the site directed mutagenesis of many conserved putative catalytic residues based on the homology with other Class I adenylyl cyclases. We will perform measurements on the synthesis of cyclic adenosine monophosphate by the wild type and mutated enzyme(s). We intend to determine the three dimensional structure of the catalytic domain.

In a project aimed at understanding the synthesis and regulation of the biosynthesis of aromatic amino acids in Mycobacterium tuberculosis, we (S. Patterson, S-K, Kim, and P. Reddy) are working on chorismate mutase, a key enzyme in the shikimate biosynthetic pathway. The results of this research may prove useful to the pharmaceutical industry for the development of new therapeutic agents against tuberculosis, particularly the drug resistant variants.

During the course of our investigations, we use the following molecular biology and biotechnology related techniques: cloning, nucleic acid (DNA and RNA) isolation, characterization by gel electrophoresis, DNA sequencing, hybridization, and gene expression measurements; protein expression, purification, and characterization by measurements in the catalytic function of enzymes; site-directed mutagenesis of proteins/enzymes to elucidate structure/function relationship.

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