title: Catalytic pathway, substrate binding and stability in SAICAR synthetase: A structure and molecular dynamics study.
abstract: The de novo purine biosynthesis is one of the highly conserved pathways among all organisms and is essential for the cell viability. A clear understanding of the enzymes in this pathway would pave way for the development of antimicrobial and anticancer drugs. Phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthetase is one of the enzymes in this pathway that catalyzes ATP dependent ligation of carboxyaminoimidazole ribotide (CAIR) with l-aspartate (ASP). Here, we describe eight crystal structures of this enzyme, in C2221 and H3 space groups, bound to various substrates and substrate mimics from a hyperthermophilic archaea Pyrococcus horikoshii along with molecular dynamics simulations of the structures with substrates. Complexes exhibit minimal deviation from its apo structure. The CAIR binding site displays a preference for pyrimidine nucleotides. In the ADP·TMP·ASP complex, the ASP binds at a position equivalent to that found in Saccharomyces cerevisiae structure (PDB: 2CNU) and thus, clears the ambiguity regarding ASP's position. A possible mode for the inhibition of the enzyme by CTP and UTP, observed earlier in the yeast enzyme, is clearly illustrated in the structures bound to CMP and UMP. The ADP.Mg(2+)·PO4·CD/MP complex having a phosphate ion between the ATP and CAIR sites strengthens one of the two probable pathways (proposed in Escherichia coli study) of catalytic mechanism and suggests the possibility of a phosphorylation taking place before the ASP's attack on CAIR. Molecular dynamic simulations of this enzyme along with its substrates at 90°C reveal the relative strengths of substrate binding, possible antagonism and the role of Mg(2+) ions.
authors.affiliation: Laboratory for Structural Biology and Biocomputing, Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Kavyashree
authors.initials: K
authors.lastname: Manjunath
authors.affiliation: Department of Bioinformatics, Alagappa University, Karaikudi 630 003, Tamil Nadu, India.
authors.forename: Jeyaraman
authors.initials: J
authors.lastname: Jeyakanthan
authors.affiliation: Laboratory for Structural Biology and Biocomputing, Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560 012, India. Electronic address: sekar@physics.iisc.ernet.in.
authors.forename: Kanagaraj
authors.initials: K
authors.lastname: Sekar
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D043902:Pyrococcus horikoshii / Q000378:metabolism
chemical_list: D010453:Peptide Synthases; C022379:phosphoribosylaminoimidazole-succinocarboxamide synthetase
keywords: ATP binding; Enzyme mechanism; Molecular dynamics; Purine de novo biosynthesis; Pyrococcus horikoshii OT3; SAICAR synthetase