Molecular structure of Escherichia coli PurT-encoded glycinamide ribonucleotide transformylase

In Escherichia colt, the PurT-encoded glycinamide ribonucleotide transformy lase, or PurT transformylase, catalyzes an alternative formylation of glyci namide ribonucleotide (GAR) in the de novo pathway for purine biosynthesis. On the basis of amino acid sequence analyses, it is known that the PurT tr ansformylase belongs to the ATP-grasp superfamily of proteins. The common t heme among members of this superfamily is a catalytic reaction mechanism th at requires ATP and proceeds through an acyl phosphate intermediate. All of the enzymes belonging to the ATP-grasp superfamily are composed of three s tructural motifs, termed the A-, B-, and C-domains, and in each case, the A TP is wedged between the B- and C-domains. Here we describe two high-resolu tion X-ray crystallographic structures of PurT transformylase from E, coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the se cond with bound AMPPNP and GAR. The latter structure is of special signific ance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in rib onucleotide binding. Specifically in PurT transformylase, the CAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363. Key amino acid side chains involved in binding the AMPPNP to the enzym e include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 Angst rom from one of the gamma-phosphoryl oxygens of the AMPPNP.