In the area of chemical bioterrorism, we (P. Reddy, S-K. Kim, G. Vasquez, D. Hancock, and K. Kole) are working to develop reference material for the rapid detection of ricin, a potent toxic protein isolated from the Castor beans. Ricin is a heterodimeric protein consisting of A chain and B chain linked by a disulfide bond. The B chain binds to galactose residues present on the cell surface glycopeptides and glycolipids and facilitates the entry of whole ricin and translocation of A chain into the cytosol. The A chain then inhibits protein synthesis by depurination of a specific adenine in the 28SrRNA which eventually leads to host cell death. We are using chemical, biochemical, and molecular biology techniques to characterize commercially available ricin.

In the area of microbial bioterrorism, we (P. Reddy and S-K. Kim) are currently 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). We use adenylyl cyclase, one of the virulence factors in Bacillus anthracis (anthrax disease), as a model system to understand its role in other bacterial pathogens. Adenylyl cyclases are classified into four major 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 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 III enzymes are regulated by a variety of small molecules such as hormones, guanosine triphosphate, and macromolecular proteins such as guanine nucleotide binding proteins, calmoduline. Class III enzymes are originally thought to be only from higher eukarytic 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 will perform the gene expression measurements and activity profiles of these 15 adenylyl cyclase genes. Yersinia pestis has a Class IV adenylyl cyclase in addition to the Class I enzyme. Class IV adenylyl cyclase is a small protein consisting of only 179 amino acids.

We (S-K, Kim and P. Reddy) have cloned the genes for Class I adenylyl cyclase (850 amino acids) and Class IV adenylyl cyclase (179 amino acids) from Yersinia pestis. The catalytic function of the Class I enzyme resides in the amino terminal half of the protein (aa1-450). The catalytic domain was expressed with histidine tag and purified in a single step. Class IV adenylyl cyclase was also expressed and purified to homogeneity. We have identified catalytic residues in both classes of the enzyme by site directed mutagenesis We will perform measurements on the synthesis of cyclic adenosine monophosphate by the wild type and mutated enzyme(s).

During the course of our investigations, we use the following molecular biology / 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.

Publications

• “Eukaryotic-like Adenylyl Cyclases in Mycobacterium tuberculosis H37Rv: Cloning and Characterization,” Sathyavelu K. Reddy, Madhavi Kamireddi, Kiran Dhanireddy, Lynn Young, Andrew Davis, and Prasad Reddy Journal of Biological Chemistry 276, 35141-35149, 2001

• “Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin,” Prasad T. Reddy, C. Rama Prasad, and P. Suryanarayana Murthy Journal of Bacteriology 185 , 5263-5268, 2003.