Publications

• Substrate-induced closing of the active site revealed by the crystal structure of pantothenate synthetase from Staphylococcus aureus.

  Satoh A., Konishi S., Tamura H., Stickland H.G., Whitney H.M., Smith A.G., Matsumura H., Inoue T.

  Pantothenate synthetase (PS, EC 6.3.2.1) is the last enzyme in the pantothenate biosynthesis pathway, a metabolic pathway identified as a potential target for new antimicrobials. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to form pantothenate. Here we report the overe ... >> More

  Pantothenate synthetase (PS, EC 6.3.2.1) is the last enzyme in the pantothenate biosynthesis pathway, a metabolic pathway identified as a potential target for new antimicrobials. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to form pantothenate. Here we report the overexpression, purification, enzyme assay, and tertiary structure of PS from Staphylococcus aureus. PS activity was experimentally confirmed, indicating a k(cat) value comparable to those of enzymes from other organisms. The structures of the apoenzyme and the reaction intermediate (pantoyl adenylate; PA) complex were determined by X-ray crystallography to resolutions of 2.5 and 1.85 A, respectively. Structural analysis indicated that the apoenzyme adopts an open and relatively mobile structure, while the complex structure is closed and entirely rigid. Structural comparison of the apoenzyme and the complex revealed how S. aureus PS undergoes open/close conformational change, and also determined the key interactions with the adenine ring of PA for a hinge bending domain closure. In the complex structure, PA and acetate are bound in the active site. We suggest that the acetate mimics the substrate beta-alanine. Therefore, the complex structure seems to represent a catalytic state poised for in-line nucleophilic attack on PA. These data also offer an alternative strategy for designing novel compounds that selectively inhibit PS activity. << Less

  Biochemistry 49:6400-6410(2010) [PubMed] [EuropePMC]

• Steady-state and pre-steady-state kinetic analysis of Mycobacterium tuberculosis pantothenate synthetase.

  Zheng R., Blanchard J.S.

  Pantothenate synthetase (EC 6.3.2.1), encoded by the panC gene, catalyzes the essential ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast and plants. Pantothenate synthetase from Mycobacterium tuberculosis was expressed in E. coli, purified to homoge ... >> More

  Pantothenate synthetase (EC 6.3.2.1), encoded by the panC gene, catalyzes the essential ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast and plants. Pantothenate synthetase from Mycobacterium tuberculosis was expressed in E. coli, purified to homogeneity, and found to be a homodimer with a subunit molecular mass of 33 kDa. Initial velocity, product, and dead-end inhibition studies showed the kinetic mechanism of pantothenate synthetase to be Bi Uni Uni Bi Ping Pong, with ATP binding followed by D-pantoate binding, release of PP(i), binding of beta-alanine, followed by the release of pantothenate and AMP. Michaelis constants were 0.13, 0.8, and 2.6 mM for D-pantoate, beta-alanine, and ATP, respectively, and the turnover number, k(cat), was 3.4 s(-1). The formation of pantoyl adenylate, suggested as a key intermediate by the kinetic mechanism, was confirmed by (31)P NMR spectroscopy of [(18)O]AMP produced from (18)O transfer using [carboxyl-(18)O]pantoate. Single-turnover reactions for the formation of pyrophosphate and pantothenate were determined using rapid quench techniques, and indicated that the two half-reactions occurred with maximum rates of 1.3 +/- 0.3 and 2.6 +/-0.3 s(-)(1), respectively, consistent with pantoyl adenylate being a kinetically competent intermediate in the pantothenate synthetase reaction. These data also suggest that both half-reactions are partially rate-limiting. Reverse isotope exchange of [(14)C]-beta-alanine into pantothenate in the presence of AMP was observed, indicating the reversible formation of the pantoyl adenylate intermediate from products. << Less

  Biochemistry 40:12904-12912(2001) [PubMed] [EuropePMC]

• A highly active pantothenate synthetase from Corynebacterium glutamicum enables the production of D-pantothenic acid with high productivity.

  Tigu F., Zhang J., Liu G., Cai Z., Li Y.

  D-Pantothenic acid (vitamin B₅) has wide applications in the feed, food, chemical, and pharmaceutical industries. Its biological production routes which employ pantothenate synthetase (PS) as the key enzyme are attractive since they avoid the tedious and time-consuming optical resolutio ... >> More

  D-Pantothenic acid (vitamin B₅) has wide applications in the feed, food, chemical, and pharmaceutical industries. Its biological production routes which employ pantothenate synthetase (PS) as the key enzyme are attractive since they avoid the tedious and time-consuming optical resolution process. However, little data is available on the activity and kinetics of this enzyme, hampering the rational selection of an efficient enzyme for the biological production of D-pantothenic acid. In this study, six phylogenetically distant PS-encoding genes, from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus thuringiensis, Bacillus cereus, and Enterobacter cloacae, were expressed in E. coli. The PS from C. glutamicum exhibited a specific activity of 205.1 U/mg and a turnover number of 127.6 s^-1, which to our best knowledge are the highest values ever reported. The addition of substrates (D-pantoic acid and β-alanine) to the E. coli strain harboring this enzyme during the early log phase of fermentation resulted in the production of 97.1 g/L of D-pantothenic acid within 32 h, corresponding to a conversion yield of 99.1% and a productivity of 3.0 g/L/h. To the best of our knowledge, this is the highest productivity reported to date. << Less

  Appl Microbiol Biotechnol 102:6039-6046(2018) [PubMed] [EuropePMC]

• Enzymological properties of pantothenate synthetase from Escherichia coli B.

  Miyatake K., Nakano Y., Kitaoka S.

  Following a previous report on physicochemical properties, the enzymological properties of a homogeneously purified preparation of pantothenate synthetase were described. The optimum pH was 10.0 and optimum temperature 30 degrees C. The lyophilized enzyme was very stable on standing at -20 degrees ... >> More

  Following a previous report on physicochemical properties, the enzymological properties of a homogeneously purified preparation of pantothenate synthetase were described. The optimum pH was 10.0 and optimum temperature 30 degrees C. The lyophilized enzyme was very stable on standing at -20 degrees C. K+ or NH4+ and Mg2+ were required as activators; other cations examined were inhibitive to various extents and the enzyme required ATP as the energy supplier. Some omega-amino acids exerted strong inhibition, and the enzyme was inhibited by some chelating agents but was not affected by SH compounds and SH inhibitors. Apparent Km for pantoate was 6.3 x 10(-5)M, for beta-alanine 1.5 x 10(-4)M, and for ATP 1.0 x 10(-4)M. According to the method of Cleland, the enzyme reaction proceeds by a Bi Uni Uni Bi Ping Pong mechanism and a scheme showing the order of binding of substrates and releasing of products is presented. << Less

  J. Nutr. Sci. Vitaminol. 24:243-253(1978) [PubMed] [EuropePMC]

• The final step of pantothenate biosynthesis in higher plants: cloning and characterization of pantothenate synthetase from Lotus japonicus and Oryza sativum (rice).

  Genschel U., Powell C.A., Abell C., Smith A.G.

  We have isolated a Lotus japonicus cDNA for pantothenate (vitamin B(5)) synthetase (PS) by functional complementation of an Escherichia coli panC mutant (AT1371). A rice (Oryza sativum) expressed sequence tag, identified by sequence similarity to PS, was also able to complement the E. coli auxotro ... >> More

  We have isolated a Lotus japonicus cDNA for pantothenate (vitamin B(5)) synthetase (PS) by functional complementation of an Escherichia coli panC mutant (AT1371). A rice (Oryza sativum) expressed sequence tag, identified by sequence similarity to PS, was also able to complement the E. coli auxotroph, as was an open reading frame from Saccharomyces cerevisiae (baker's yeast). The Lotus and rice cDNAs encode proteins of approx. 34 kDa, which are 65% similar at the amino acid level and do not appear to encode N-terminal extensions by comparison with PS sequences from other organisms. Furthermore, analysis of genomic sequence flanking the coding sequence for PS in Lotus suggests the original cDNA is full-length. The Lotus and rice PSs are therefore likely to be cytosolic. Southern analysis of Lotus genomic DNA indicates that there is a single gene for PS. Recombinant PS from Lotus, overexpressed in E. coli AT1371, is a dimer. The enzyme requires d-pantoate, beta-alanine and ATP for activity and has a higher affinity for pantoate (K(m) 45 microM) than for beta-alanine (K(m) 990 microM). Uncompetitive substrate inhibition becomes significant at pantoate concentrations above 1 mM. The enzyme displays optimal activity at about 0.5 mM pantoate (k(cat) 0.63 s(-1)) and at pH 7.8. Neither oxopantoate nor pantoyl-lactone can replace pantoate as substrate. Antibodies raised against recombinant PS detected a band of 34 kDa in Western blots of Lotus proteins from both roots and leaves. The implications of these findings for pantothenate biosynthesis in plants are discussed. << Less

  Biochem. J. 341:669-678(1999) [PubMed] [EuropePMC]

• Pantothenate synthetase is essential but not limiting for pantothenate biosynthesis in Arabidopsis.

  Jonczyk R., Ronconi S., Rychlik M., Genschel U.

  Pantothenate (vitamin B5) is the universal precursor for coenzyme A (CoA), an essential cofactor that is required in the metabolism of carbohydrates and fatty acids. The final step of bacterial and eukaryotic pantothenate biosynthesis is catalyzed by pantothenate synthetase (PTS), which is encoded ... >> More

  Pantothenate (vitamin B5) is the universal precursor for coenzyme A (CoA), an essential cofactor that is required in the metabolism of carbohydrates and fatty acids. The final step of bacterial and eukaryotic pantothenate biosynthesis is catalyzed by pantothenate synthetase (PTS), which is encoded by a single gene in Arabidopsis thaliana (AtPTS). There was debate whether PTS represents the only mode of pantothenate production because previous biochemical evidence pointed to an additional pantothenate pathway in plants. Here we show that insertional mutant alleles of AtPTS confer a recessive embryo-lethal phenotype with mutant embryos arrested at the preglobular stage. Exogenous pantothenate was required for normal seed development and germination and also facilitated the remaining life cycle. Complementation of the mutant phenotype was likewise achieved by heterologous expression of E. coli PTS (panC). The panC transgene increased the total PTS activity in leaves by up to 500-fold but did not affect the steady-state level of pantothenate, indicating that PTS is essential but not limiting for pantothenate production. The auxotrophic AtPTS knockout phenotype suggests that the embryo and possibly all other tissues are autonomous for the biosynthesis of pantothenate. This view is consistent with the near-ubiquitous expression of AtPTS as judged by promoter:beta-glucuronidase analysis. Given the high demand for CoA during storage oil accumulation, we analyzed transcript and metabolite patterns of CoA biosynthesis in seeds. The data indicate that the pantothenate and CoA contents follow distinct developmental programs and that both transcriptional and posttranslational control mechanisms are important for CoA homeostasis. << Less

  Plant Mol. Biol. 66:1-14(2008) [PubMed] [EuropePMC]