Carboxylation of phenylphosphate by phenol carboxylase, an enzyme system of anaerobic phenol metabolism.

Several lines of evidence indicate that the first step in the anaerobic metabolism of phenol is phenol carboxylation to 4-hydroxybenzoate; this reaction is considered a biological Kolbe-Schmitt carboxylation. A phenol carboxylase system was characterized by using a denitrifying Pseudomonas strain, K 172, which catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate. The enzymatic isotope exchange activity (100 nmol min-1 mg-1 of protein) requires Mn2+ and K+. We show that this system also catalyzes the carboxylation of phenylphosphate (the phosphoric acid monophenyl ester) to 4-hydroxybenzoate and phosphate. The specific activity of phenylphosphate carboxylation at the optimal pH of 6.5 is 12 nmol of CO2 fixed min-1 mg-1 of protein. Phenylphosphate cannot be replaced by Mg(2+)-ATP and phenol. The carboxylase activity requires Mn2+ but, in contrast to the isotope exchange activity, does not require K+. The apparent Km values are 1.5 mM dissolved CO2 and 0.2 mM phenylphosphate. Several convenient assays for phenylophosphate carboxylation are described. The isotope exchange reaction and the net carboxylation reaction are catalyzed by the same oxygen-sensitive enzyme, which has a half-life in an air-saturated solution of less than 1 min. Both activities cochromatographed with a protein with a Mr of 280,000, and both activities were induced only after anaerobic growth on phenol. The carboxylation of phenylphosphate suggests that phenylphosphate itself is the physiological CO2 acceptor molecular of this novel CO2 fixation reaction. Alternatively, phenylphosphate could simulate the unknown natural precursor. It is suggested that the formation of an enzyme-bound phenolate anion from the activated phenolic compound is the rate-determining step in the carboxylation reaction.     

Regulation of the operon encoding ribonucleotide reductase: role of the negative sites in nrd repression.

Expression of the nrd genes was previously shown to be controlled by both positive and negative regulation (C. K. Tuggle and J. A. Fuchs, EMBO J. 5:1077-1085, 1986). Two regions, one located 5' and one located 3' of the nrd promoter (nrdP), were identified as negative regulatory sites since deletion of these sequences increased nrd expression. These regions of DNA have sequence similarities, and a looping mechanism was proposed to explain the requirement for two distinct sites in nrd repression. To investigate the role of these sequences in regulating nrd, a gel electrophoresis assay was used to detect the proteins that bind to the nrd regulatory sites. A protein that bound to restriction fragments containing the negative regulatory sites but not to other DNA fragments was identified in cell extracts and was partially purified. DNase I footprinting experiments showed that the binding protein protects the 5' negative site previously identified in vivo. The 3' negative site also identified in vivo was not required in vitro for high-affinity protein binding to the 5' site, but lower-affinity binding to this site could be detected. Specific binding to the 5' site was found to be elevated approximately 10-fold in crude extracts from thymine-starved cells as compared with that in extracts from unstarved cells. This higher activity was also evident in purified preparations, suggesting that thymine starvation increases the expression of the negative regulatory protein. The finding that a purified protein preparation binds both negative regulatory sites indicates that this preparation contains the nrd repressor protein or proteins. Insertion of 37 base pairs (3.5 helix turns) of DNA at a HpaII site or 35 base pairs (3.3 turns) at a MnlI site between the 5' regulatory sites and nrdP abolished the increase in nrd expression resulting from thymine starvation in vivo, but negative regulation appeared to be less affected than when either negative site was deleted. Insertion of DNA in these constructs was shown not to affect repressor binding in vitro, indicating either that a simple model of DNA looping to bring equivalent operator sites into physical proximity does not explain repression at nrd or that the distance between sites is sufficient that helical turns are of little importance.     

Nuclease-coated bacteriophage: a sensitive tool for studying antigenic reactivity of synthetic sequence fragments

Staphylococcal nuclease was conjugated to bacteriophage T₄ using glutaraldehyde as a cross-linking agent. The conjugated phage is inactivated by antinuclease antibodies and this inactivation is specifically inhibited by nuclease in concentrations as low as 10^?9m. Two fragments of the enzyme, namely P₂ (residues 6–48) and P₃ (residues 49–149) inhibit to a much lower extent the inactivation of the conjugated bacteriophage by the antibodies, than the native enzyme, but when mixed together (forming the noncovalent complex, nuclease-T), the inhibition curve obtained is similar to that obtained by native nuclease. This sensitive system was applied for testing different synthetic sequence fragments and for studying the complementarity of synthetic sequences in the P₂ region with native P₃.     

The synthesis of 5-amino-2,6-anhydro-5-deoxy-D-glycero-D-gulo-Heptonic acid and its polycondensation to oligomers

5-Amino-2,6-anhydro-5-deoxy-D-glycero-D-gulo-heptonic acid has been synthesized by conventional introduction of an amino function via azide displacement, starting with a suitable derivative of 2,6-anhydro-D-glycero-L-manno-heptonic acid. The amino acid was converted into the methyl ester hydrochloride which, in methanolic sodium methoxide, gave oligomeric and polymeric amides, depending on the conditions applied. Four oligomeric esters, as well as the corresponding N-(2,4-dinitrophenyl) derivatives of the amino acids, could be separated by paper chromatography. The oligomers could be saponified under mild, basic conditions.     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

The binding of ⁴⁵Ca²⁺ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg²⁺ (no ATP) binding was half-maximal at 1.4 · 10^?6M Ca²⁺ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 10⁶ M^?1) and the Hill coefficient was 1.0. In the absence of Mg²⁺, binding was half-maximal at 3 · 10^?7 M Ca²⁺ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 10⁷ and 2 · 10⁶ M^?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg²⁺ binding to about half of the total Ca²⁺ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites.     

Carbon assimilation pathways in sulfate-reducing bacteria II. Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus

The strict anaerobe Desulfobacter hydrogenophilus is able to grow autotrophically with CO₂, H₂, and sulfate as sole carbon and energy sources. The generation time at 30°C under autotrophic conditions in a pure mineral medium was 15 h, the growth yield was 8 g cell dry mass per mol sulfate reduced to H₂S. Enzymes of the autotrophic CO₂ assimilation pathway were investigated. Key enzymes of the Calvin cycle and of the acetyl CoA pathway could not be found. All enzymes of a reductive citric acid cycle were present at specific activities sufficient to account for the observed growth rate. Notably, an ATP-citrate lyase (1.3 mol · min^-1 · mg cell protein^-1) was present both in autotrophically and in heterotrophically grown cells, which was rapidly inactivated in the absence of ATP. The data indicate that in D. hydrogenophilus a reductive citric acid cycle is operating in autotrophic CO₂ fixation. Since other autotrophic sulfate reducers possess an acetyl CoA pathway for CO₂ fixation, two different autotrophic pathways occur in the same physiological group.Dedicated to Prof. H. G. Wood on the occasion of his 80th birthday     

Juvenile hormone and 20-hydroxyecdysone as primary and secondary stimuli of vitellogenesis in Aedes aegypti

Female Aedes aegypti that were fed blood and immediately abdominally ligated did not deposit yolk. Injection of 20-hydroxyecdysone (1.5–5.0 ng) or topical application of juvenile hormone (JH) analogue methoprene (25 pg) did not induce vitellogenesis in these abdomens. When blood-gorged ligated abdomens were treated with both hormones, however, vitellogenesis was stimulated in 60% of treated animals. Rocket immunoelectrophoresis indicated that vitellin concentration per follicle in treated animals was similar to that in intact controls. When ligated abdomens were first treated with methoprene and immediately injected with a crude head extract of egg development neurosecretory hormone, vitellogenin synthesis was induced at a rate similar to that in blood-fed controls. Methoprene at this concentration (25 pg), did not cause an increase in whole-body ecdysteroid titers. Larger amounts of methoprene (1.65 ng) were needed to stimulate egg development and ecdysteroid production. Implantation of ecdysone-secreting ovaries into ligated abdomens did not stimulate vitellogenesis in the recipients. However, in recipients that were first treated with methoprene (25 pg), implantation of ecdysone-secreting ovaries resulted in normal egg development. These experiments indicate that the appearance of JH precedes 20-hydroxyecdysone in stimulating vitellogenesis following blood feeding in Ae. aegypti.     

Introduction and establishment ofApanteles flavipes [Hym.: Braconidae] onDiatraea saccharalis [Lep.: Pyralidae] in texas

The braconid parasite,Apanteles flavipes (Cameron), was introduced into the Lower Rio Grande Valley of Texas in 1977 and has become established onDiatraea saccharalis (F.) attacking 4 species of host plants. Approximately 71% of the 26,971 adult parasites released were released in sugarcane,Saccharum officinarum L., with the remainder being released on field corn,Zea mays L., broomcorn,Sorghum vulgare technicum (Koern.) and Johnson grass,Sorghum halepense (L.). Recoveries indicate dispersal of at least 4 km from release sites.     

Preliminary crystallographic investigations of two phycobiliproteins.

Single crystal x-ray diffraction investigations are in progress on two phycobiliproteins. C-phycocyanin from Anabaena variabilis crystallizes in space group P63 with a = b = 154 A and c = 40 A. The crystallographic asymmetric unit is (alphabeta)2 with a total molecular mass of 7.0-10(4) daltons. B-phycoerythrin from Porphyridium cruentum crystallizes in space group R3 with a = b = 189 A and c = 60 A. This molecule has the unusual molecular stoichiometry (alphabeta)6gamma and the crystallographic asymmetric unit is (alphabeta)2gamma1/3. This requires that the gamma chain undergo a perfect 3-fold disordering about the crystallographic 3 axis, i.e. the gamma chain must occupy three symmetry-equivalent positions, each with an occupancy of one-third.