3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and the four-carbon monosaccharide D-erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus is a member of the DAH7PS Ibeta subfamily, which also includes the KDO8PS enzymes. KDO8PS (3-deoxy-D-manno-octulosonate-8-phosphate synthase) catalyzes a closely related reaction of PEP with the five-carbon monosaccharide D-arabinose 5-phosphate (A5P). DAH7PS from P. furiosus requires a metal ion for activity and, unlike other characterized DAH7PS enzymes, is not inhibited by aromatic amino acids. Purified P. furiosus DAH7PS is able to utilize not only the four-carbon phosphorylated monosaccharides E4P and 2-deoxy-D-erythrose 4-phosphate but also the five-carbon phosphorylated monosaccharides A5P, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate with similar kcat but much increased KM values. DL-glyceraldehyde 3-phosphate and D-glucose 6-phosphate are not substrates. The structure of recombinant P. furiosus DAH7PS in complex with PEP was determined to 2.25 A resolution. The asymmetric unit consists of a dimer of (beta/alpha)8-barrel subunits. Analysis of the buried surfaces formed by dimerization and tetramerization, as observed in the crystal structure, provides insight into both the oligomeric status in solution and the substrate ambiguity of P. furiosus DAH7PS. P. furiosus DAH7PS is both the first archaeal and the first "naked" DAH7PS (without N-terminal extensions) to be fully characterized functionally and structurally. The broad substrate specificity of this DAH7PS, the lack of allosteric inhibition, and various structural features indicate that, of the enzymes characterized to date, P. furiosus DAH7PS may be the contemporary protein closest to the ancestral type I enzyme.