DHQ1 is a class I aldolase enzyme that catalyzes the reversible dehydration of 3-dehydroquinic acid to form 3-dehydroshikimic acid by multi-step mechanism that involves the formation of Schiff base species’. DHQ1 is present in plants and several bacterial sources, such as Escherichia coli, Salmonella typhi and Staphylococcus aureus, but it does not have any counterpart in human cells. It has been suggested that DHQ1 may act as a virulence factor in vivo as the deletion of the aroD gene, which encodes DHQ1, has been proven to afford satisfactory live oral vaccines. This fact has identified DHQ1 as a promising target in the search for new anti-virulence agents to combat widespread antibiotic resistance, which has become one of the most important public health issues of the early 21st century.
Two epoxides were designed as irreversible inhibitors of the DHQ1 enzyme and to study the binding requirements of the linkage to the enzyme. We have described the first crystal structure of DHQ1 from S. typhi (St-DHQ1) covalently modified by epoxide S at 1.4 Å (Scheme 1). Surprisingly, the crystal structure reveals that the ligand is covalently attached to the enzyme as a Schiff base and additional chemical modifications took also place.
Scheme 1. Crystal structure of the St-DHQ1 covalently modified by epoxide S and proposed mechanism.
We then have developed the first example of an ammonium derivative that causes a specific modification of the active site of DHQ1 (Scheme 2). The resolution at 1.35 Å of the crystal structure of DHQ1 from Salmonella typhi chemically modified by this ammonium derivative revealed that the ligand is covalently attached to the essential Lys170 through the formation of an amine. Moreover, in vitro anti-virulence studies showed that the reported compound is able to reduce the ability of Salmonella Enteritidis to kill A459 respiratory cells.
Scheme 2. Crystal structure of the St-DHQ1 covalently modified by an ammonium derivative and proposed mechanism.