Structural Analysis of the Active Site Geometry of N⁵-Carboxyaminoimidazole Ribonucleotide Synthetase from Escherichia coli^,

Abstract

N 5 -carboxyaminoimidazole ribonucleotide synthetase (N 5 -CAIR synthetase) converts 5-aminoimidazole ribonucleotide (AIR), MgATP, and bicarbonate into N 5 -CAIR, MgADP, and P i . The enzyme is required for de novo purine biosynthesis in microbes yet is not found in humans suggesting that it represents an ideal and unexplored target for antimicrobial drug design. Here we report the x-ray structures of N 5 -CAIR synthetase from Escherichia coli with either MgATP or MgADP/P i bound in the active site cleft. These structures, determined to 1.6-Å resolution, provide detailed information regarding the active site geometry before and after ATP hydrolysis. In both structures, two magnesium ions are observed. Each of these is octahedrally coordinated, and the carboxylate side chain of Glu 238 bridges them. For the structure of the MgADP/P i complex, crystals were grown in the presence of AIR and MgATP. No electron density was observed for AIR, and the electron density corresponding to the nucleotide clearly revealed the presence of ADP and P i rather than ATP. The bound P i shifts by approximately 3 Å relative to the γ-phosphoryl group of ATP and forms electrostatic interactions with the side chains of Arg 242 and His 244. Since the reaction mechanism of N 5 -CAIR synthetase is believed to proceed via a carboxyphosphate intermediate, we propose that the location of the inorganic phosphate represents the binding site for stabilization of this reactive species. Using the information derived from the two structures reported here, coupled with molecular modeling, we propose a catalytic mechanism for N 5 -CAIR synthetase.The original pathway proposed for de novo purine biosynthesis by Buchanan in the late 1950s involved the 10-step conversion of phosphoribosyl pyrophosphate into the common branchpoint intermediate inosine monophosphate (1). This view of purine biosynthesis existed well into the mid-1990s until it was discovered that the 10-step pathway was not universal across all organisms (2-4). Indeed, de novo purine biosynthesis is different in microbes versus humans, and the difference centers around the synthesis of the key intermediate, 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). In bacteria and yeast, CAIR is produced from 5-aminoimidazole ribonucleotide (AIR) by the action of two enzymes rather than one as in higher eukaryotes (Scheme 1). Clearly, de novo purine biosynthesis has evolutionarily diverged among lower and higher organisms. This divergent nature of a primary metabolic pathway has significant ramifications for the design of novel antimicrobial agents. Genetic studies have shown that both N 5 -CAIR synthetase and N 5 -CAIR mutase are required for microbial growth (6-9). Deletion of either enzyme results in a purine auxotroph that is unable to propagate in human or mouse serum and is not viable in animal models predictive of disease (6,(10)(11)(12). These results support the conclusion that inhibitors of these enzymes could be potential antibacterial and antifungal agents.The first structural ...

The enzymes of biotin dependent CO2 metabolism: What structures reveal about their reaction mechanisms

“…In contrast, magnesium (B) is coordinated by the side chain carboxylate of Glu 274 (a bidentate ligand), oxygen atoms from the a-and c-phosphoryl groups, a water molecule, and a carboxylate oxygen contributed by Glu 288. The coordination geometry exhibited by the S. aureus biotin carboxylase has also been observed in the biotin carboxylase domain of pyruvate carboxylase 16 and in other enzymes belonging to the ATP-grasp superfamily including carbamoyl phosphate synthetase, 42 glycinamide ribonucleotide transformylase, 36 and N 5 -carboxyaminoimidazole ribonucleotide synthetase, 43,44 among others.…”

“…Rather bicarbonate, in its protonated form, reacts with ATP to generate a carboxyphosphate intermediate, which adopts a pseudochain conformation as suggested by molecular mechanics calculations. 43 According to the proposed mechanism, the carboxyphosphate decomposes to CO 2 via an intramolecular proton abstraction by the phosphate group. 44 Recently, the Tong group reported a structure of E. coli biotin carboxylase with two ADP molecules bound in the active site.…”

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“…(3). The diagram shows two sites of N 5 -Carboxyaminoimidazole Ribonucleotide Synthetase from Escherichia coli (see Ref [32] and PDB structure code 3ETH Ref [3]). The ATP molecules are represented color tube style whereas the Mg(II) cations are depicted as green balls.…”

Communication: Near edge x-ray absorption fine structure spectroscopy of aqueous adenosine triphosphate at the carbon and nitrogen K-edges

“…1 ATPutilizing enzymes have special pockets or grooves in their three-dimensional structures to bind the ATP and catalyze the loss of the phosphate, usually in the presence of a metal cocatalyst, such as magnesium ͑Mg 2+ ͒. [2][3][4] To reliably model the uptake of ATP into these special binding sites, more accurate models of solvated ATP, as well as the protein crystal structures, are needed. This requires an understanding of the details of the solvation environment and how that environment is disrupted by association with proteins.…”