A novel GAR transformylase has been isolated and characterized from E. coli. The protein, a product of the purT gene, is a monomer of molecular weight 42 kDa and catalyzes the production of beta-formyl GAR from formate, ATP, and beta-GAR. As such it is an alternative to the formyl-folate utilizing purN GAR transformylase. No significant homology exists between the two transformylases. However, the purT protein shows significant homology to the purK protein, also involved in purine biosynthesis. Two different purT reactions have been characterized: one producing fGAR from ATP, beta-GAR, and formate and the other producing acetyl phosphate and ADP from acetate and ATP. The purT GAR transformylase is the first unknown de novo purine biosynthetic enzyme to be discovered in the last 30 years and represents another step forward in understanding cellular control of purine levels.