The C-3 deoxygenation step in the biosynthesis of d-forosamine (4-N,N-dimethylamino-2,3,4,6-tetradeoxy-d-threo-hexopyranose), a constituent of spinosyn produced by Saccharopolyspora spinosa, was investigated. The spnQ gene, proposed to encode a TDP-4-keto-2,6-dideoxy-d-glucose 3-dehydratase was cloned and overexpressed in E. coli. Characterization of the purified enzyme established that it is a PMP and iron-sulfur containing enzyme which catalyzes the C-3 deoxygenation in a reductase-dependent manner similar to that of the previously well characterized hexose 3-dehydrase E1 from Yersinia pseudotuberculosis. However, unlike E1, which has evolved to work with a specific reductase partner present in its gene cluster, SpnQ lacks a specific reductase, and works efficiently with general cellular reductases ferredoxin/ferredoxin reductase or flavodoxin/flavodoxin reductase. SpnQ also catalyzes C-4 transamination in the absence of an electron transfer intermediary and in the presence of PLP and l-glutamate. Under the same conditions, both E1 and the related hexose 3-dehydrase, ColD, catalyze C-3 deoxygenation. Thus, SpnQ possesses important features which distinguish it from other well studied homologues, suggesting unique evolutionary pathways for each of the three hexose 3-dehydrases studied thus far.