Ribose-5-phosphate isomerase (Rpi) acts as a key enzyme in the oxidative and reductive pentose-phosphate pathways for the conversion of ribose-5-phosphate (R5P) to ribulose-5-phosphate and vice versa. We have determined the crystal structures of Rpi from Thermus thermophilus HB8 in complex with the open chain form of the substrate R5P and the open chain form of the C2 epimeric inhibitor arabinose-5-phosphate as well as the apo form at high resolution. The crystal structures of both complexes revealed that these ring-opened epimers are bound in the active site in a mirror symmetry binding mode. The O1 atoms are stabilized by an oxyanion hole composed of the backbone amide nitrogens in the conserved motif. In the structure of the Rpi⅐R5P complex, the conversion moiety O1-C1-C2-O2 in cis-configuration interacts with the carboxyl oxygens of Glu-108 in a water-excluded environment. Furthermore, the C2 hydroxyl group is presumed to be highly polarized by short hydrogen bonding with the side chain of Lys-99. R5P bound as the ring-opened reaction intermediate clarified the high stereoselectivity of the catalysis and is consistent with an aldose-ketose conversion by Rpi that proceeds via a cis-enediolate intermediate.Ribose-5-phosphate isomerase (Rpi 1 ; EC 5.3.1.6) is ubiquitous throughout all living cells and highly conserved in amino acid sequences. Rpi acts as a key enzyme in the oxidative pentose-phosphate cycle where it catalyzes the reversible conversion of ribose-5-phosphate (R5P) to ribulose-5-phosphate (Ru5P). Rpi additionally plays a central role in the reductive pentose-phosphate cycle (Calvin cycle) of photosynthetic organisms. Rpi catalyzes the final step of the conversion of glucose-6-phosphate into ribose-5-phosphate, which is required for the synthesis of nucleotides. It also converts R5P to Ru5P in the final step to regenerate ribulose-1,5-bisphosphate as the acceptor of CO 2 in the Calvin cycle. In the non-oxidative pathway of the pentose-phosphate cycle, R5P is the precursor of 5-phosphoribosylpyrophosphate, which is utilized for syntheses of amino acids such as histidine and tryptophan, purine and pyrimidine nucleotides, and NAD (1), whereas Ru5P is the riboflavin precursor (2). In the Calvin cycle, it has been shown that Rpi forms a functional multienzyme complex with five other enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), to catalyze the consecutive reactions on chloroplast thylakoid membranes in situ (3). The direction of the reaction catalyzed by Rpi is essentially driven by the R5P and Ru5P concentrations.The catalytic mechanism of Rpi (Fig. 1A) is believed to initiate with a ring opening of the substrate followed by isomerization of the open chain form. Isotope exchange studies have suggested that the isomerization between ketose and aldose by triosephosphate isomerase (TIM), phosphoglucose isomerase (PGI), and Rpi involves a proton transfer between the C1 and C2 positions of the substrate via the cis-enediol(ate) intermediate (4, 5). The transferred proton in c...
Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyzes the rate-limiting step in coenzyme A (CoA) biosynthesis by transferring an adenylyl group from ATP to 4'-phosphopantetheine (Ppant), yielding 3'-dephospho-CoA (dPCoA). The crystal structure of PPAT from Thermus thermophilus HB8 (Tt PPAT) complexed with Ppant has been determined by the molecular-replacement method at 1.5 A resolution. The overall fold of the enzyme is almost the same as that of Escherichia coli PPAT, a hexamer having point group 32. The asymmetric unit of Tt PPAT contains a monomer and the crystallographic triad and dyad coincide with the threefold and twofold axes of the hexamer, respectively. Most of the important atoms surrounding the active site in E. coli PPAT are conserved in Tt PPAT, indicating similarities in their substrate binding and enzymatic reaction. The notable difference between E. coli PPAT and Tt PPAT is the simultaneous substrate recognition by all six subunits of Tt PPAT compared with substrate recognition by only three subunits in E. coli PPAT. Comparative analysis also revealed that the higher stability of Tt PPAT arises from stabilization of each subunit by hydrophobic effects, hydrogen bonds and entropic effects.
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