Untitled

Abstract

ATIC, the product of the purH gene, is a 64 kDa bifunctional enzyme that possesses the final two activities in de novo purine biosynthesis, AICAR transformylase and IMP cyclohydrolase. The crystal structure of avian ATIC has been determined to 1.75 A resolution by the MAD method using a Se-methionine modified enzyme. ATIC forms an intertwined dimer with an extensive interface of approximately 5,000 A(2) per monomer. Each monomer is composed of two novel, separate functional domains. The N-terminal domain (up to residue 199) is responsible for the IMPCH activity, whereas the AICAR Tfase activity resides in the C-terminal domain (200-593). The active sites of the IMPCH and AICAR Tfase domains are approximately 50 A apart, with no structural evidence of a tunnel connecting the two active sites. The crystal structure of ATIC provides a framework to probe both catalytic mechanisms and to design specific inhibitors for use in cancer chemotherapy and inflammation.

Crystal Structures of Human Bifunctional Enzyme Aminoimidazole-4-carboxamide Ribonucleotide Transformylase/IMP Cyclohydrolase in Complex with Potent Sulfonyl-containing Antifolates

“…For the BW1540 complex, the rotation and translation search, using all data from 15.0 -3.5 Å, identified two monomers in the asymmetric unit with a final correlation co-efficient and R cryst of 55.5 and 44.3%, respectively (note that for the first noise peak, correlation coefficient ϭ 26.4 and R cryst ϭ 56.2%). The two monomers form the biologically relevant homodimer as observed in previous avian ATIC structures (5)(6)(7). For the BW2315 complex, the rotation search (15.0 -4.0 Å) identified four major solutions with correlation co-efficients of 13.9 -12.1 (noise peak ϭ 8.8); the subsequent translational search yielded a final correlation co-efficient of 60.8 and a R cryst of 39.4%.…”

“…. Interestingly, this Asn 431Ј interaction with the AICAR 5-amino group was not observed in previous structures where its side chain pointed away from the active site (5)(6)(7)22). Otherwise, additional active site water molecules are observed in the BW1540 structure that interact with the AICAR phosphate and sugar (Fig.…”

“…1B). XMP is carried through protein purification and crystallization and also selectively binds to only one of the two IMP cyclohydrolase active sites in the homodimer in apo avian and human ATIC structures (5,22,36).…”

“…1B) in which the transformylase and cyclohydrolase active sites were separated by ϳ50 Å. However, no evidence of channeling or tunneling of 5-formyl-AICAR between the two domains has been forthcoming (5,8), although strong electropositive patches that connect the two active sites may sequester 5-formyl-AICAR to the vicinity of the binding sites.…”

A Methanocaldococcus jannaschii Archaeal Signature Gene Encodes for a 5-Formaminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-Monophosphate Synthetase

“…The product of this first reaction is then cyclized by the IMP cyclohydrolase activity that resides in the amino terminus of the enzyme (1). The two-domain structure of PurH has recently been confirmed by the x-ray crystal structure of the avian enzyme (2).…”