Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily^,

Abstract

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. Small PurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the apo form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analog, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. Availability of a ternary complex enabled mapping of the active site thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. A surprising discovery, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculations about the regulatory role of the auxiliary site in PurLSQ complex formation as well as the evolutionary relationship of PurL's from different organisms.The purine biosynthetic pathway is ubiquitous in most living organisms, and it is a ten-step process for converting phosphoribosyl pyrophosphate to inosine monophosphate. Formyl glycinamide ribonucleotide amidotransferase (FGAR-AT), also known by its gene product † This work was supported by the NIH grants RR15301 and GM073220 (SEE). SEE is indebted to the W. M. Keck Foundation and the Lucille P. Markey Charitable Trust. AAH was supported by a NSF predoctoral fellowship. AAH and JS were supported by NIH Grant GM32191. The Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems at MIT is supported by grants (NSF-0070319 and NIH GM68762). Supporting Information Available A table of primers used in TmPurL cloning and mutagenesis, a table summarizing the SV-AUC results, the Coomassie-stained gels for StPurL and mutants, a graph of the distribution of species obtained after SEDFIT analysis of SV-AUC data for wild type and mutant TmPurL, a topology diagram of TmPurL, circular dichroism spectra for wild type and mutant TmPurL, and an elution profile of wild type TmPurL after incubation with radioactive ATP. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 2. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript name, PurL, catalyzes the fourth...

Structure of [NiFe] Hydrogenase Maturation Protein HypE from Escherichia coli and Its Interaction with HypF

“…However, the structure of D. vulgaris HypE was determined in complex with ATP and confirmed the predicted location of its binding site. Also, the recent crystal structures of PurL have revealed a molecule of bound ADP (1) or ATP (31). All of these structures show a common ATP binding location.…”

“…However, Mg 2ϩ ions are present in the enzyme-ATP complex, indicating that the binding of ATP occurs with the participation of Mg 2ϩ . Two such ions coordinated by the phosphate groups of ATP and protein side chains were observed in HypE Dv (45) and in PurL complexes (31). The side chains that coordinate Mg 2ϩ ions are structurally conserved in HypE and PurL.…”

“…Despite low sequence identity, the fold of HypE is similar to that of PurM (PDB ID 1CLI [25]), PurL (PDB ID 2HRU [31]), and ThiL (PDB ID 1VQV [S. Eswaramoorthy and S. Swaminathan, unpublished data]). Structural superposition of HypE on PurM and ThiL suggests that the chain assignment of the N-terminal regions (residues 1 to 32) of both subunits in ThiL should be swapped to obtain the correct dimer.…”

Structure of an N-terminally truncated selenophosphate synthetase fromAquifex aeolicus

“…A structural homology analysis with the DALI program (Holm & Sander, 1998) indicated that the protein fold is the typical PurM-like fold (Table 2). SPS-ÁN shares high structural similarities with aminoimidazole ribonucleotide synthetase (PurM; Li et al, 1999), formylglycinamide ribonucleotide amidotransferase (PurL; Anand et al, 2004;Morar et al, 2006), thiamine-monophosphate kinase (ThiL) and [NiFe] hydrogenase maturation protein (HypE; Watanabe et al, 2007), which are members of the PurM superfamily, with Z scores of 15.3-26.2 and r.m.s.d.s of 2.5-3.9 Å , despite the low levels of sequence identity (10-20%; Table 2). …”

“…It is intriguing that some of the bacterial SPSs, all known archaeal SPSs and eukaryal SPS2 species are themselves Sec-containing proteins (selenoproteins), in which a catalytically important Cys residue in the N-terminal segment is replaced by Sec (Fleischmann et al, 1995;Low et al, 1995;Bult et al, 1996). According to sequence similarity, the SPS proteins are classified as members of the PurM superfamily (Anand et al, 2004;Morar et al, 2006). The selenophosphate synthesized by SPS is also utilized in the sulfur/selenium-exchange reaction to obtain the 2-selenouridine modification, such as 5-methylaminomethyl-2-selenouridine (mnm 5 Se 2 U) in tRNA anticodons, from the 2-thiouridine modification (Leinfelder et al, 1990;Veres et al, 1990Veres et al, , 1992.…”