Publications

• Structure and kinetics of indole-3-glycerol phosphate synthase from <i>Pseudomonas aeruginosa</i>: Decarboxylation is not essential for indole formation.

  Soderholm A., Newton M.S., Patrick W.M., Selmer M.

  In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGPS) starts with a condensation step in which the substrate's carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation that res ... >> More

  In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGPS) starts with a condensation step in which the substrate's carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation that restores the aromaticity of the phenyl. IGPS from <i>Pseudomonas aeruginosa</i> has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. Since the 1960s, this step has been considered to be mechanistically essential based on studies of the IGPS-phosphoribosylanthranilate isomerase fusion protein from <i>Escherichia coli</i> Here, we present the crystal structure of <i>P. aeruginosa</i> IGPS in complex with reduced CdRP, a nonreactive substrate analog, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate, with an ∼1000× lower rate of IGP formation than from the native substrate. We also show that <i>E. coli</i> IGPS, at an even lower rate, can produce IGP from decarboxylated substrate. Our structure of <i>P. aeruginosa</i> IGPS has eight molecules in the asymmetric unit, of which seven contain ligand and one displays a previously unobserved conformation closer to the reactive state. One of the few nonconserved active-site residues, Phe²⁰¹ in <i>P. aeruginosa</i> IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that despite IGPS's classification as a carboxy-lyase (<i>i.e.</i> decarboxylase), decarboxylation is not a completely essential step in its catalysis. << Less

  J Biol Chem 295:15948-15956(2020) [PubMed] [EuropePMC]

• Isolation of cDNAs encoding the tryptophan pathway enzyme indole-3-glycerol phosphate synthase from Arabidopsis thaliana.

  Li J., Chen S., Zhu L., Last R.L.

  Plant Physiol. 108:877-878(1995) [PubMed] [EuropePMC]

• Indole-3-glycerol phosphate synthetase of Escherichia coli, an enzyme of the tryptophan operon.

  Creighton T.E., Yanofsky C.

  J Biol Chem 241:4616-4624(1966) [PubMed] [EuropePMC]

• Extremely stable indole-3-glycerol-phosphate synthase from hyperthermophilic archaeon Pyrococcus furiosus.

  Arif M., Rashid N., Perveen S., Bashir Q., Akhtar M.

  The gene-encoding Indole-3-glycerol phosphate synthase, a key enzyme involved in the cyclization of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate, from Pyrococcus furiosus was cloned and expressed in Escherichia coli. The gene product was produced in the soluble and active form. The recombi ... >> More

  The gene-encoding Indole-3-glycerol phosphate synthase, a key enzyme involved in the cyclization of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate, from Pyrococcus furiosus was cloned and expressed in Escherichia coli. The gene product was produced in the soluble and active form. The recombinant protein, purified to apparent homogeneity, displayed highest activity at 100 °C and pH of 5.5. The recombinant enzyme followed Michaelis-Menten kinetics exhibiting apparent V_max and K_m values of 20 ± 0.5 μmol min^-1 mg^-1 and 140 ± 10 µM, respectively. The activation energy, determined from the linear Arrhenius plot, was 17 ± 0.5 kJ mol^-1. A unique property of PfInGPS is its stability against denaturants and temperature. There was no significant change in activity even in the presence of 8 M urea or 5 M guanidine hydrochloride. Furthermore, recombinant PfInGPS was highly thermostable with a half-life of 200 min at 100 °C. To the best of our knowledge, this is the most stable indole-3-glycerol phosphate synthase characterized to date. << Less

  Extremophiles 23:69-77(2019) [PubMed] [EuropePMC]