Structural and Mutational Analysis of Substrate Complexation by Anthranilate Phosphoribosyltransferase from Sulfolobus solfataricus

Abstract

The metabolic synthesis and degradation of essential nucleotide compounds are primarily carried out by phosphoribosyltransferases (PRT) and nucleoside phosphorylases (NP), respectively. Despite the resemblance of their reactions, five classes of PRTs and NPs exist, where anthranilate PRT (AnPRT) constitutes the only evolutionary link between synthesis and degradation processes. We have characterized the active site of dimeric AnPRT from Sulfolobus solfataricus by elucidating crystal structures of the wild-type enzyme complexed to its two natural substrates anthranilate and 5-phosphoribosyl-1-pyrophosphate/Mg 2؉ . These bind into two different domains within each protomer and are brought together during catalysis by rotational domain motions as shown by small angle x-ray scattering data. Steady-state kinetics of mutated AnPRT variants address the role of active site residues in binding and catalysis. Results allow the comparative analysis of PRT and pyrimidine NP families and expose related structural motifs involved in nucleotide/ nucleoside recognition by these enzyme families.Nucleotide compounds are central to the production of genetic material, the amino acids histidine and tryptophan, and cofactors such as NAD. Their metabolism is largely based on the reversible transfer of a phosphoribosyl group to aromatic bases. Although forward and reverse ribosylation processes share a close resemblance (Fig. 1), they are carried out by different enzymes and in the context of distinct metabolic pathways. Synthesis reactions are performed by phosphoribosyltransferases (PRT). 3 These use PRPP as universal phosphoribosyl donor. These reactions, which are dependent on metal ions, involve the displacement of the 1-pyrophosphate group of PRPP and formation of a N-1Ј-glycosidic bond to a nitrogenated base specific for each PRT. Conversely, the release of aromatic bases from nucleotides involves first the production of a nucleoside intermediate by nucleotidases or phosphatases, followed by the cleavage of the glycosidic bond by either nucleoside phosphorylases (NP) or nucleoside hydrolases. NP enzymes are key to nucleotide salvage both in prokaryotes and in eukaryotes. They catalyze the reversible phosphorolysis of the glycosidic bond of nucleosides to yield free bases and ribose-1-phosphate.Despite the chemical resemblance of the compounds intervening in synthesis and degradation reactions by PRTs and NPs ( Fig. 1), respectively, the enzymes involved in these catalyses lack structural similarity. Three diverse PRT classes that act on aromatic bases are known to date, as follows: (i) those with a canonical fold classified as type PRT-I, which includes most of the PRTs for which an atomic structure is available (1); (ii) type PRT-II comprising quinolate PRT (2-3) and nicotinate PRT (4); and (iii) anthranilate PRT (AnPRT) (5-7) (Fig. 1A). NPs are also classified in two groups with distinct folds as follows: (i) NP-I, which act primarily on purine nucleosides but also accept the pyrimidine uridine, and (ii) NP-II, which degrade t...

A Rationally Designed Monomeric Variant of Anthranilate Phosphoribosyltransferase from Sulfolobus solfataricus is as Active as the Dimeric Wild-type Enzyme but Less Thermostable

“…This structure, in combination with mutational analysis, made it possible to identify the role of individual residues for substrate binding at the active site of each subunit. 20 To test the significance of homo-dimer formation for the function and extreme thermostability of ssAnPRT, 21 we exchanged two residues at the contact interface individually and in combination to loosen the dimer. The double mutant formed a pure monomer with wild-type catalytic activity but reduced thermal stability.…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…Among the differences in PRPP binding between the two enzymes are the binding properties of Asp-Glu, which bind to MG2 and to the oxygen of C-5 (Asp residue only) in S. solfataricus anthranilate phosphoribosyltransferase. MG2 furthermore coordinates to the 5-phosphate and to the oxygens of C-4 and C-5 of ribose 5-phosphate (289). The corresponding aspartate and gluatmate residues of the M. tuberculosis enzyme are coordinated to MG1 (Glu252) or to MG2 (Asp251).…”

“…Interestingly, in S. solfataricus anthranilate phosphoribosyltransferase Gly79 also forms a hydrogen bond to the anthranilate nitrogen. Thus, this residue may be particularly important in positioning anthranilate and PRPP for inline attack of anthranilate on C-1 of PRPP (289).…”

YbiB from Escherichia coli, the Defining Member of the Novel TrpD2 Family of Prokaryotic DNA-binding Proteins

“…The reactions of the TrpD and NP-II enzymes resemble each other chemically; TrpD synthesizes a glycosidic bond, whereas NP-II enzymes phosphorolyze it. Despite sharing a low sequence similarity (on average 12% sequence identity) and displaying some significant differences, TrpD and NP-II proteins are both members of the phosphoribosyl transferase class III superfamily and have a similar fold, their active sites share common features, and both enzymes have a similar quaternary structure, where homodimers are formed by head-to-head interactions of equivalent secondary structure elements (2,3).…”

“…This enzyme, from the metabolic pathway of tryptophan biosynthesis, catalyzes the condensation of the nitrogenated base anthranilate and the phosphoribosyl donor 5Ј-phosphoribosyl-␣1-pyrophosphate (PRPP). 4 Crystal structures of TrpD have been solved (2)(3)(4)(5), and its physicochemical and catalytic properties have been studied in detail (6 -9). The other homologue to TrpD2 (on average 10% sequence identity) is the NP-II (10 -13).…”