Katherine Hoffmann

Research


Bacterial pathogens must scavenge iron from their host for growth and proliferation during infection.  They have evolved several strategies to do this, one being the biosynthesis and excretion of small, high-affinity iron chelators known as siderophores.  Two general pathways for siderophore biosynthesis exist: the well-characterized nonribosomal peptide synthetase (NRPS)-dependent pathway and the NRPS-independent siderophore (NIS) pathway, which relies on a different family of understudied and novel synthetases.  NIS synthesis enzymes fall into at least three distinct families (A, B, and C,) based on substrate specificity, and with few exceptions are associated with some of the most virulent and persistent bacterial infections (staph, anthrax, plague) We are initially interested in structurally characterizing this understudied family of enzymes, as the single example of structurally characterized NIS synthetase (a type A enzyme) has described a novel-binding fold and enzyme chemistry.  Further structural information, both within type A and new structures of types B and C, as well as clear information about the secondary substrate binding pocket, and higher oligomerization state, are the primary goals for our work.

 

Since NIS sythetases fall into three distinct families, we have identified several members of each subtype, and propose a parallel approach to cloning, expression and structural characterization. Beginning with two identified siderophore synthesis pathways, we will structurally characterize the type A enzyme DesD from Streptomyces coelicolor, and from Bacillus anthraxis we will explore the type A NIS synthetase AsbA and the type C AsbB structures, from the petrobactin siderophore biosynthesis pathway (an obligate siderophore associated with anthrax pathogenesis). We will compare our AsbA, AsbB structures with the structures of enzymes out of Yersina pestis (types A and C), and Francisella tularensis (type B), all of which were identified using bioinformatics.  Investigation of these siderophore biosynthetic pathways will provide novel structural information for uncharacterized subtypes of NIS synthetases, which we intend to use to begin a long-term program exploring the kinetic and chemical activity potentially leading to therapeutics for bacterial infection, but also to the better understanding of new enzyme chemistries.  

 

Currently all projects in the lab are in cloning stages, or are waiting for materials to begin cloning. As the genes are cloned into overexpression vectors, we will individually begin expression, purification and solubility tests to maximize efficient expression, and then progress to crystallization trials.