X-ray crystal structure of aminoimidazole ribonucleotide synthetase (PurM), from the Escherichia coli purine biosynthetic pathway at 2.5 A resolution.
BACKGROUND
The purine biosynthetic pathway in procaryotes enlists eleven enzymes, six of which use ATP. Enzymes 5 and 6 of this pathway, formylglycinamide ribonucleotide (FGAR) amidotransferase (PurL) and aminoimidazole ribonucleotide (AIR) synthetase (PurM) utilize ATP to activate the oxygen of an amide within their substrate toward nucleophilic attack by a nitrogen. AIR synthetase uses the product of PurL, formylglycinamidine ribonucleotide (FGAM) and ATP to make AIR, ADP and P(i).


RESULTS
The structure of a hexahistidine-tagged PurM has been solved by multiwavelength anomalous diffraction phasing techniques using protein containing 28 selenomethionines per asymmetric unit. The final model of PurM consists of two crystallographically independent dimers and four sulfates. The overall R factor at 2.5 A resolution is 19.2%, with an R(free) of 26.4%. The active site, identified in part by conserved residues, is proposed to be a long groove generated by the interaction of two monomers. A search of the sequence databases suggests that the ATP-binding sites between PurM and PurL may be structurally conserved.


CONCLUSIONS
The first structure of a new class of ATP-binding enzyme, PurM, has been solved and a model for the active site has been proposed. The structure is unprecedented, with an extensive and unusual sheet-mediated intersubunit interaction defining the active-site grooves. Sequence searches suggest that two successive enzymes in the purine biosynthetic pathway, proposed to use similar chemistries, will have similar ATP-binding domains.
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Department of Chemistry and Chemical Biology Cornell University Ithaca, New York 14853, USA.
Kappock
T J
TJ
Stubbe
J
J
Weaver
T M
TM
Ealick
S E
SE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins; D008025:Ligases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011687:Purines / Q000096:biosynthesis*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013431:Sulfates / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011687:Purines; D013431:Sulfates; D000255:Adenosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C022380:phosphoribosylaminoimidazole synthase; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; C102675:PurL protein, E coli