Flagellin inhibits Myoviridae phage phiCTX infection of Pseudomonas aeruginosa strain GuA18: purification and mapping of binding site.

PhiCTX is a double-stranded DNA phage of the Myoviridae family that converts Pseudomonas aeruginosa into a cytotoxin producer. A 42-kDa phiCTX-inhibiting protein was purified from the outer membrane fraction of P. aeruginosa strain GuA18 by octyl-beta-glucoside extraction, DEAE-chromatography, and mono-Q HPLC. This protein had an isoelectric point of 5.4 and bound specifically [125I]-labeled phiCTX. The N-terminal amino acid sequence of six out of seven Lys-C fragments was highly similar (87%) to that of the entire of type-a flagellin of P. aeruginosa strain PAK. At a concentration of 14 nM, purified flagellin protein caused a 50% decrease in the phage titer after a 20-min incubation at 37 degrees C (PhI50). The presence of ethanol was necessary to reconstitute the inhibitory activity. In contrast, no ethanol treatment was necessary for the inhibitory activity of the sheared flagellin filaments from P. aeruginosa strain GuA18, which consists of the 42-kDa flagellin subunits and the synthesized 17-mer phage-binding-peptide NGSNSDSERTALNGEAK, representing flagellin residues 100-116 of P. aeruginosa strain PAK. The PhI50 was 10 nM and 200 nM, respectively. Antisera against the flagellin filament protein as well as against the 17-mer peptide neutralized phage infection. These results indicated that the amino acid region 100-116 of the flagellin subunit of strain GuA18 is involved in phiCTX binding. This region might play a role in phage attachment.

Identification of the bile acid-binding site of the ileal lipid-binding protein by photoaffinity labeling, matrix-assisted laser desorption ionization-mass spectrometry, and NMR structure.

The ileal lipid-binding protein (ILBP) is the only physiologically relevant bile acid-binding protein in the cytosol of ileocytes. To identify the bile acid-binding site(s) of ILBP, recombinant rabbit ILBP photolabeled with 3-azi- and 7-azi-derivatives of cholyltaurine was analyzed by a combination of enzymatic fragmentation, gel electrophoresis, and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. The attachment site of the 3-position of cholyltaurine was localized to the amino acid triplet His(100)-Thr(101)-Ser(102) using the photoreactive 3,3-azo-derivative of cholyltaurine. With the corresponding 7,7-azo-derivative, the attachment point of the 7-position could be localized to the C-terminal part (position 112-128) as well as to the N-terminal part suggesting more than one binding site for bile acids. By chemical modification and NMR structure of ILBP, arginine residue 122 was identified as the probable contact point for the negatively charged side chain of cholyltaurine. Consequently, bile acids bind to ILBP with the steroid nucleus deep inside the protein cavity and the negatively charged side chain near the entry portal. The combination of photoaffinity labeling, enzymatic fragmentation, MALDI-mass spectrometry, and NMR structure was successfully used to determine the topology of bile acid binding to ILBP.

A new esterase for the cleavage of pivalic acid-containing prodrug esters of cephalosporins.

An extracellular esterase from the actinomycetes Amycolatopsis orientalis was found by screening. It is capable of splitting the isomeric mixture (K/J) of (I, Scheme 1) into 7-amino-3-methoxymethyl-3-cephem-4-carboxylic acid, pivalic acid, and acetaldehyde with a high yield. The purified enzyme of 55.4 Kd by SDS-PAGE shows an N-terminal sequence of VRTCADLVRTYDLPGAVTH. The isoelectric point is 8.9 +/- 0.1. It can be immobilized with good yield to VA-Epoxy Biosynth. Besides the above-mentioned reaction, the esterase cleaves many other esters such as methyl-2-chloropropionic acid.

[Degradation of antipyrin by pyrazon-degrading bacteria (author's transl)]. / Abbau von Antipyrin durch Pyrazon-abbauende Bakterien.

Bacteria with the ability to grow on pyrazon as sole source of carbon were isolated from soil. They also are able to grow on antipyrin. Then three metabolites of antipyrin can be isolated from the culture fluid which were identified as 2,3-dimethyl-1-(cis-2,3-dihydro-2,3-dihydroxy-4,6-cyclohexadiene-1-yl)-pyrazolone (5) (I), as 2,3-dimethyl-1-(2,3-dihydroxyphenyl)-pyrazolone (5) (II) and as 2,3-dimethyl-pyrazolone (5) (III), respectively. Compound I and II were used as substrates for enzyme studies. A dioxygenase catalyzes the enzymatic conversion of antipyrin into compound I. In the presence of NAD as cosubstrate compound I is transformed into compound II by a dehydrogenase. A pure preparation of metapyrocatechase from pyrazon-degrading bacteria converts compound II into the dephenylated heterocyclic moiety of antipyrin (III) and into 2-pyrone-6-carboxylic acid. Based on the results of the enzymatic studies a pathway for the degradation of antipyrin is proposed.

Purification and properties of pyrazon dioxygenase from pyrazon-degrading bacteria.

Chromatography on DEAE-cellulose and gel filtration on Sephadex revealed that pyrazon dioxygenase from pyrazon-degrading bacteria consists of three different enzyme components. No component alone oxidizes the phenyl moiety of pyrazon, only when the three components are combined can oxidation be detected. Following electron paramagnetic resonance and ultraviolet measurements the protein nature of the three components was determined: component A1 (molecular weight about 180000,red-brown in colour) is an iron-sulphur protein. The existence of approximately two moles of iron and two moles of inorganic sulphur per mole of protein was demonstrated. This enzyme component was purified to homogeneity in disc electrophoresis. Component A2 is a yellow protein of a molecular weight of about 67000. FAD was shown to be the prosthetic group of this protein. Component B (molecular weight about 12000, brown in colour) is a protein of the ferredoxin type, which was purified to homogeneity, as demonstrated by disc electrophoresis. A hypothetical scheme for the cooperation of the three components is proposed: component A2 accepts as cosubstrate NADH and functions as a ferredoxin reductase. The ferredoxin, component B, has the function of an electron carrier. The conversion of the substrates is effected by component A1, the terminal dioxygenase.

[Breakdown of the herbicide pyramin by micro-organisms of the soil (author's transl)]. / Der Abbau des Herbizids Pyramin durch Mikroorganismen des Bodens

The herbicide Pyramin, which is employed in the cultivation of beets to combat broad-leaf weeds, contains the herbicidal substance 5-amino-4-chloro-2-phenyl-3 (2H) pyridazinone, abbreviated pyrazone. The breakdown of pyrazone in the soil was investigated and it was found that this substance disappears relatively quickly and that the dephenylated heterocycle of pyrazone 5-amino-4-chloro-3 (2H) pyridazinone is obtained as transformation product. It was possible to isolate bacteria, which grow on pyrazone as the only carbon source, from soil samples originating from different parts of the world. Four compounds are excreted during the cultivation of pyrazone-degrading bacteria in a pyrazone mineral salt medium. With the aid of the structure of these metabolites and enzymatic tests, a scheme for the bacterial breakdown of pyrazone is proposed.

[On the enzymatics of the microbial breakdown of pyrazone (author's transl)]. / Zur Enzymatik des mikrobiellen Pyrazonabbaus

From samples of earth taken in different parts of the world bacteria were isolated which grow on pyrazone as the only source of carbon. When these bacteria are grown in a pyrazone mineral salt medium, four compounds are excreted into the medium. The structures of these compounds furnish information on the catabolic route of pyrazone. Since the suggested scheme of breakdown was incomplete, enzymatic tests were carried out to clarify the matter. It was possible to carry out the first steps of breakdown also in the cell-free extract of the pyrazone-degrading bacteria. For the second step of pyrazone breakdown, 2 different enzymes of the same catalytic activity were identified. For the oxidative cleavage of the pyrocatechole derivative 2 different enzymes were found: an ortho- and a meta-clearing enzyme. The 2-hydroxy muconic acid decarboxylase was identified as a further enzyme. The importance of this enzyme is discussed in connection with the further breakdown.