Abstract: The crystal structure of the class IV adenylyl cyclase (AC) from Yersinia pestis (Yp) is reported at 1.9 A resolution. The class IV AC fold is distinct from the previously described folds for class II and class III ACs. The dimeric AC-IV folds into an antiparallel eight-stranded barrel whose connectivity has been seen in only three previous structures: yeast RNA triphosphatase and two proteins of unknown function from Pyrococcus furiosus and Vibrio parahaemolyticus. Eight highly conserved ionic residues E10, E12, K14, R63, K76, K111, D126, and E136 lie in the barrel core and form the likely binding sites for substrate and divalent cations. A phosphate ion is observed bound to R63, K76, K111, and R113 near the center of the conserved cluster. Unlike the AC-II and AC-III active sites that utilize two-Asp motifs for cation binding, the AC-IV active site is relatively enriched in glutamate and features an ExE motif as its most conserved element. Homologs of Y. pestis AC-IV, including human thiamine triphosphatase, span the three kingdoms of life and delineate an ancient family of phosphonucleotide processing enzymes.