The ATP-grasp enzymes consist of a superfamily of 21 proteins that contain an atypical ATP-binding site, called the ATP-grasp fold. The ATP-grasp fold is comprised of two α + β domains that “grasp” a molecule of ATP between them and members of the family typically have an overall structural design containing 3 common conserved focal domains. The founding members of the family consist of biotin carboxylase, D-ala-D-ala ligase and glutathione synthetase, all of which catalyze the ATP-assisted reaction of a carboxylic acid with a nucleophile via the formation of an acylphosphate intermediate. While most members of the superfamily follow this mechanistic pathway, studies have demonstrated that two enzymes catalyze only the phosphoryl transfer step and thus are kinases instead of ligases. Members of the ATP-grasp superfamily are found in several metabolic pathways including de novo purine biosynthesis, gluconeogenesis and fatty acid synthesis. Given the critical nature of these enzymes, researchers have actively sought the development of potent inhibitors of several members of the superfamily as antibacterial and anti-obseity agents. In this review, we will discuss the structure, function, mechanism and inhibition of the ATP-grasp enzymes.

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A comparative investigation of the substrate requirements for the enzyme 5-aminoimidazole ribonucleotide (AIR) carboxylase from E. coli and G. gallus has been conducted using in vivo and in vitro studies. In Escherichia coli, two enzymes PurK and PurE are required for the transformation of AIR to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). The Gallus gallus PurCE is a bifunctional enzyme containing AIR carboxylase and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synthetase. The E. coli PurE and the C-terminal domain of the G. gallus PurCE protein maintain a significant degree of amino acid sequence identity and also share CAIR as a product of their enzymatic activities. The substrate requirements of AIR carboxylases from E. coli and G. gallus have been compared by a series of in vitro experiments. The carbamic acid, N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) is a substrate for the E. coli PurE (Mueller et al., 1994) but not for the G. gallus AIR carboxylase. In contrast, AIR and CO2 are substrates for the G. gallus AIR carboxylase. The recognition properties of the two proteins were also compared using inhibition studies with 4-nitro-5- aminoimidazole ribonucleotide (NAIR). NAIR is a tight-binding inhibitor of the G. gallus AIR carboxylase (K(i) = 0.34 nM) but only a steady-state inhibitor (K(i) = 0.5 microM) of the E. coli PurE. These data suggest significant differences in the transition states for the reactions catalyzed by these two evolutionarily related enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Two successive steps in de novo purine biosynthesis are catalyzed by the enzymes 5-aminoimidazole ribonucleotide (AIR) carboxylase and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synthetase. Amino acid sequence alignments of the proteins from various sources suggested that several unusual differences exist within the structure and function of these enzymes. In vertebrates, a bifunctional enzyme (PurCE) catalyzes successive carboxylation and aspartylation steps of AIR to form SAICAR. This is in contrast to the three proteins, PurK, PurE, and PurC, from Escherichia coli which have recently been shown to require 2 equiv of ATP for the AIR to SAICAR conversion in the presence of physiological HCO3- concentrations (Meyer et al., 1992). A comparative study of these proteins has been initiated using a high-production, heterologous expression system for the Gallus gallus AIR carboxylase-SAICAR synthetase and yields purified enzyme following a two-step procedure. Selective assays have been developed for all the enzymatic activities of the bifunctional protein. The G. gallus AIR carboxylase has no ATP dependence and displays a Km for HCO3- that is 10-fold lower than that for the related PurE protein from E. coli, supporting the hypothesis that the two enzymes require different substrates. No common cofactors or metals are required for catalysis. Each catalytic activity has been shown to be independent by selective inactivation of SAICAR synthetase with the affinity agent 5'-[4-(fluorosulfonyl)benzoyl]-adenosine (FSBA) and inhibition of AIR carboxylase with a tight-binding inhibitor 4-nitro-5-aminoimidazole ribonucleotide (NAIR). The native protein aggregates, and limited proteolysis indicates that the global structure of the protein involves two independent folding domains, each containing a different catalytic site.

The increase in the incidence of both hospital- and community-acquired antibiotic-resistant infections is a major concern to the healthcare community. There have been only two new classes of antibiotics approved by the FDA over the past 40 years, and clearly there is a growing need for additional antimicrobial agents. In this paper, we present our work on the discovery of a class of benzophenone containing compounds that possess good activity against MRSA, VISA, VRSA, and VRE and moderate activity against E. coli. These compounds display MIC values in the 0.5-2.0 mg/L range and are not cytotoxic against mammalian cells. Extensive structure-activity relationship studies revealed that the benzophenone was absolutely essential for antibacterial activity as was the presence of a cationic group. Although these agents display DNA binding activity, we observed that these compounds do not inhibit any macromolecular synthesis reliant upon DNA nor do they inhibit lipid or cell wall biosynthesis. Instead, we found that these agents cause membrane depolarization, indicating that the bacterial membrane was the primary site of action for these agents. Our studies suggest that caution should be taken in assigning the mechanism of action for DNA binding antibiotics.

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 ...

PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of glycinamide ribonucleotide through a catalytic mechanism requiring Mg 2؉ ATP and formate. From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase from Escherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains. In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B-and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding. Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg 2؉ ATP, Mg 2؉ -5-adenylylimidodiphosphate, Mg 2؉ -␤,␥-methyleneadenosine 5-triphosphate, Mg 2 ؉ ATP␥S, or Mg 2؉ ADP. Taken together, these studies indicate that the conformation of the so-called "T-loop," delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket. This sensitivity to nucleotide identity is in sharp contrast to that observed for the "P-loop"-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.