Structural and functional studies of Salmonella effector proteins

Gram-negative bacteria of the Salmonella enterica species are ubiquitous facultative intracellular pathogens one of the most infective in humans, causing diseases from gastroenteritis to typhoid fever. Salmonella secretes a range of proteins called effectors to gain entry and colonize the host cell. These effectors are secreted by type 3 secretion system. Upon endocytic internalization by the host cell the bacterium resides in a membrane-bound compartment - the Salmonella containing vacuole (SCV). The effector proteins prevent conversion of SCV into lysosomes and promote bacterial survival and replication within this compartment. The function of effectors varies from interfering protein synthesis and host cell signaling pathways, mediating vesicle traffic to rearranging actin cytoskeleton. We have undertaken studies of several effectors from Salmonella enterica serovar Typhimurium, such as SopD2, GtgE and SpvB, to understand their mechanism of action at the molecular level. We have expressed and purified these proteins and undertaken their crystallization. We will present our most recent results.

Structural studies of NenD1, an argininosuccinate lyase involved in the biosynthesis of nenemycin

Nenemycin is a newly discovered 31-membered cyclodepsipeptide antibiotic isolated from an aquatic Streptomyces. This cyclic molecule contains ester and peptide bonds and is biosynthesized by the combined action of multiple enzymes. Among those is NenD1, an argininosuccinate lyase involved in the synthesis of 2S,3R--2,3-diaminobutanoic acid (DABA), a modified amino acid later added to the cycle of the nenemycin biosynthesis. The structure of NenD1 has been determined at  2.7 Å, showing the homotetrameric arrangement of the enzyme. A new crystal form was obtained after reductive methylation of lysine residues and the resulted 2.9 Å structure reveals features not present in the original. The active site is located at the interface of three subunits and its architecture was compared with other similar enzymes. Our study provides further insights into the synthesis mechanism of the unusual amino acid.

Structure-function studies of CrmK, an oxidase involved in Caerulomycin A biosynthesis

Caerulomycin A (CRM A) is an immunosupressive agent that has a unique 2,2'-bipyridine core structure. Isolated from a marine-derived Actinoalloteichus cyanogriseus, this natural product exhibits antifungal, anti-amoebic, antitumor, and antimicrobial activities. Its biosynthetic pathway consists of more than 20 enzymes, at least seven of which are putatively involved in post-PKS/NRPS modifications of the scaffold. Among these, CrmK is a flavin-dependent oxidase. We have determined the crystal structure of CrmK bound to its flavin adenin dinucleotide (FAD) cofactor at 1.9 Å resolution. FAD linkage to CrmK is observed via two covalent bonds with protein residues His64 and Cys124. This crystal structure, combined with the activity analysis of both wild-type CrmK and a series of mutants, has revealed the role of active site residues lining the substrate and FAD binding pocket. Our studies add additional molecular insights into the structure and function relationship of the bicovalently flavinylated oxidases.