L-tyrosine is the precursor of PQQ biosynthesis in Hyphomicrobium X

A method was developed to study amino acids as possible precursors of PQQ biosynthesis. Cultures of Hyphomicrobium X, growing on [13C]methanol, were supplemented with unlabelled amino acids. Uptake and participation in metabolism were determined via gas chromatography/mass spectrometry of derivatized amino acids, obtained from hydrolyzed cellular protein, by measuring their 12C content. Several amino acids appeared to be incorporated into the protein to a significant extent, without degradation or conversion. Among these were the aromatic amino acids, L-tyrosine and L-phenylalanine. Using the same replacement approach, their incorporation into PQQ was determined by 1H- and 13C-NMR spectroscopy of purified PQQ obtained from the culture medium. It appeared that the complete carbon skeleton of tyrosine was present, forming the o-quinone and pyrrole-2-carboxylic acid moieties in PQQ, while phenylalanine was not incorporated at all. Starting with L-tyrosine, possible biosynthetic routes to PQQ are discussed.     

A search for intermediates in the bacterial biosynthesis of PQQ

Studies on the biosynthesis of pyrroloquinoline quinone (PQQ) were performed with Acinetobacter calcoaceticus PQQ- -mutants belonging to genetically different complementation groups. All mutants were unable to grow on L-arabinose, the conversion of this substrate by the organism only occurring via membrane-bound quinoprotein (PQQ-containing) glucose dehydrogenase. In general, the same observation and conclusion applied to shikimate and quinate, requiring active quinoprotein quinate dehydrogenase (EC 1.1.99.--), although some mutants appeared to be leaky with respect to PQQ biosynthesis under this condition. A number of mutants were unable to grow on anthranilate and accumulated this compound when the growth medium was supplemented with L-kynurenine. Combined with other observations, it strongly suggests that these are deletion mutants, missing a gene for synthesis of anthranilate hydroxylase (EC 1.14.12.1) as well as nearby located genes for the biosynthesis of PQQ. Supplementation of the growth media with amino acids did not result in stimulation of PQQ biosynthesis. Also cross-feeding experiments, using normal and permeabilized cells with extensive variation in combination and conditions, resulted in neither stimulation nor reconstitution of PQQ synthesis. Under conditions optimal for PQQ production in the wild-type strain, as well as under stress conditions using a limiting amount of added cofactor, excretion of intermediates by PQQ- -mutants could not be detected. Similar results were obtained with PQQ- -mutants from Methylobacterium organophilum and Pseudomonas aureofaciens. A tentative explanation, accounting for the absence of detectable intermediates in the biosynthetic route, is given.     

Competitive potentiation of acetylcholine effects on neuronal nicotinic receptors by acetylcholinesterase-inhibiting drugs

The effects of the acetylcholinesterase inhibitors physostigmine and tacrine on alpha4beta2 and alpha4beta4 subtypes of neuronal nicotinic acetylcholine (ACh) receptors, expressed in Xenopus laevis oocytes, have been investigated. In voltage-clamp experiments low concentrations of physostigmine and tacrine potentiate ion currents induced by low concentrations of ACh, whereas at high concentrations they inhibit ACh-induced ion currents. These dual effects result in bell-shaped concentration-effect curves. Physostigmine and tacrine, by themselves, do not act as nicotinic receptor againsts. The larger potentiation is observed with 10 microM: physostigmine on alpha4beta4 nicotinic receptors and amounts to 70% at 1 microM: ACh. The mechanism underlying the effects of physostigmine on alpha4beta4 ACh receptors has been investigated in detail. Potentiation of ACh-induced ion current by low concentrations of physostigmine is surmounted at elevated concentrations of ACh, indicating that this is a competitive effect. Conversely, inhibition of ACh-induced ion current by high concentrations of physostigmine is not surmounted at high concentrations of ACh, and this effect appears mainly due to noncompetitive, voltage-dependent ion channel block. Radioligand binding experiments demonstrating displacement of the nicotinic receptor agonist (125)I-epibatidine from its recognition sites on alpha4beta4 ACh receptors by physostigmine confirm that physostigmine is a competitive ligand at these receptors. A two-site equilibrium receptor occupation model, combined with noncompetitive ion channel block, accounts for the dual effects of physostigmine and tacrine on ACh-induced ion currents. It is concluded that these acetylcholinesterase-inhibiting drugs interact with the ACh recognition sites and are coagonists of ACh on alpha4-containing nicotinic ACh receptors.     

Temperature- and structure-dependent interaction of pyrethroids with the sodium channels in frog node of Ranvier

(1) The interaction of a series of pyrethroid insecticides with the Na+ channels in myelinated nerve fibres of the clawed frog, Xenopus laevis, was investigated using the voltage clamp technique. (2) Out of 11 pyrethroids 9 insecticidally active compounds induce a slowly decaying Na+ tail current on termination of a step depolarization, whereas the Na+ current during depolarization was hardly affected. These tail currents are most readily explained by a selective reduction of the rate of closing of the activation gate in a fraction of the Na+ channels that have opened during depolarization. (3) The rate of decay of the Na+ tail current varies considerably with pyrethroid structure. After alpha-cyano pyrethroids the decay is at least one order of magnitude slower than after non-cyano pyrethroids. The decay always follows a single-exponential time course and is reversibly slowed when the temperature is lowered from 25 to 0 degrees C. Arrhenius plots in this temperature range are linear. (4) These results indicate that the relaxation of the activation gate in pyrethroid-affected Na+ channels is governed by an apparent first order, unimolecular process and that the rate of relaxation is limited by a single energy barrier. Application of transition state theory shows that after alpha-cyano pyrethroids this energy barrier is 9.6 kJ/mol higher than after non-cyano pyrethroids. (5) Differences in rate of decay of the Na+ tail current account for the reported differences in repetitive nerve activity induced by various pyrethroids. In addition, the effect of temperature on the rate of decay explains the increase in repetitive activity with cooling.     

Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.     

Determination of the number of cross-links in a protein gel from its mechanical and swelling properties

The relation between the chemical structure of a protein and the physical properties of a heat-set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress-strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross-links in the gel. The cross-links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross-links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory-Huggins lattice model, can also be used to calculate the number of cros-links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross-links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.