DL-a-Monofluoromethylputrescine is a potent irreversible inhibitor of Escherichia coli ornithine decarboxylase.

DL-alpha-Monofluoromethylputrescine (compound R.M.I. 71864) is an enzyme-activated irreversible inhibitor of the biosynthetic enzyme ornithine decarboxylase from Escherichia coli. This compound, however, has much less effect in vitro on ornithine decarboxylase obtained from Pseudomonas aeruginosa. These findings are in contrast with those previously found with the substrate analogue DL-alpha-difluoromethylornithine (compound R.M.I. 71782). The K1 of the DL-alpha-monofluoromethylputrescine for the E. coli ornithine decarboxylase is 110 microM, and the half-life (t1/2) calculated for an infinite concentration of inhibitor is 2.1 min. When DL-alpha-monofluoromethylputrescine is used in combination with DL-alpha-difluoromethylarginine (R.M.I. 71897), an irreversible inhibitor of arginine decarboxylase, in vivo in E. coli, both decarboxylase activities are inhibited (greater than 95%) but putrescine levels are only decreased to about one-third of control values and spermidine levels are slightly increased.     

Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme

The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized "anonymous" genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases. This set of unique, apparently species-specific, sequences are referred to as ORFans. The biochemical and structural analysis of ORFan gene products is of both evolutionary and functional interest. Here we report the cloning and expression of Escherichia coli ORFan ykfE gene and the functional characterization of the encoded protein. Under physiological conditions, the protein is a homodimer with a strong affinity for C-type lysozyme, as revealed by co-purification and co-crystallization. Activity measurements and fluorescence studies demonstrated that the YkfE gene product is a potent C-type lysozyme inhibitor (K(i) approximately 1 nm). To denote this newly assigned function, ykfE has now been registered under the new gene name Ivy (inhibitor of vertebrate lysozyme) at the E. coli genetic stock center.     

Glycosylation of Escherichia coli L-asparaginase.

Reductive coupling with sodium cyanoborhydride has been used with lactose and N-acetylneuraminyl lactose to prepare glycosylated Escherichia coli L-asparaginase. A substantial degree of modification can be achieved without significant loss of enzyme activity. The lactosylated enzyme shows increased thermal stability and resistance to proteolytic cleavage and is cleared more rapidly from the plasma of mice, compared to native asparaginase. The effect on clearance varies directly with the degree of lactosylation. Asparaginase modified with N-acetylneuraminyl lactose, in contrast, with approximately 13.6 mol of N-acetylneuraminyl lactose/mol of enzyme, is cleared more slowly, with a t 1/2 that is approximately twice that of the native enzyme.     

L-asparaginase synthesis by Escherichia coli B

We have studied the influence of strain of organism, temperature, and medium on the production of the antileukemic intracellular enzyme L-asparaginase by E. coli B grown in shaken flasks. Five strains of E. coli B exhibited wide differences in their capacities to synthesize the EC-2 form of L-asparaginase active against leukemia. For the most productive strain, when grown in a casein hydrolysate medium, maximal production of L-asparaginase occurred at 25°C. At this temperature, the organism required glycerol, glucose, or other mono-saccharides to synthesize L-asparaginase. Synthesis was stimulated when glycerol was used in place of glucose, but not in its presence. The effect of glycerol on L-asparaginase synthesis was most evident when the cells were grown at 37°C, rather than at 25°C. With 0.25% glucose, cells had a specific activity of 409 I.U./g; with glycerol cells had a specific activity of 553 I.U./g. At 25°C, both cell and L-asparaginase synthesis were increased by the use of 0.25% glycerol resulting in only a slight increase in specific activity of the cells. The addition of zinc, copper, manganese, iron, L-asparagine, L-glutamine, or L-aspartic acid had no effect on L-asparaginase synthesis in the casein hydrolysate medium. L-aspartic acid (10^?2 M) enhanced L-asparaginase synthesis in a synthetic medium that lacked these metals or L-asparagine, L-glutamine, or L-aspartic acid; cells grown under these conditions had a specific activity of 90 I.U./g. In the casein hydrolysate medium, cell morphology was correlated with temperature of incubation.     

Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases

A protein capable of inhibiting trypsin and other pancreatic proteases has been purified to homogeneity from Escherichia coli by conventional procedures and affinity chromatography. It is stable for at least 30 min at 100 degrees C and pH 1.0, but it is inactivated by digestion with pepsin. The inhibitor has an apparent molecular weight of 38,000 as determined by gel filtration and must be a homodimer since it contains a single 18,000-dalton subunit upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor has an isoelectric point of 6.1. One dimeric molecule of the inhibitor can bind two trypsin molecules to form a mixed tetrameric complex, in which trypsin molecules are completely inhibited. The inhibitor is not digested by the trypsin. When N-benzoyl-DL-arginine-p-nitroanilide was used as a trypsin substrate, half-maximal inhibition was observed at 22 nM. This protein also inhibits chymotrypsin, pancreatic elastase, rat mast cell chymase, and human serosal urokinase, but it does not inhibit human pulmonary tryptase, kallikrein, papain, pepsin, Staphylococcus aureus V8 protease, subtilisin, and thermolysin. Surprisingly, it did not inhibit any of the eight soluble endoproteases recently isolated from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, and Pi) nor the chymotrypsin-like (protease I) and trypsin-like (protease II) esterases in E. coli. The inhibitor is localized to the periplasmic space and its level did not change with different growth media or stages of cell growth. The physiological function of this E. coli trypsin inhibitor is unknown. We suggest that E. coli trypsin inhibitor be named "Ecotin."     

The primary structure of L-asparaginase from Escherichia coli

The carboxymethylated L-asparaginase from Escherichia coli A-1--3 was fragmented with cyanogen bromide and the resulting peptides were isolated by using gel filtration on Sephadex G-50 and column chromatography on DE-52. The amino acid sequences of the 7 cyanogen bromide peptides thus obtained were established completely or partially by further fragmentation with trypsin, chymotrypsin and pepsin, and the Dansyl Edman method. Based on the above results and the complete sequences of the tryptic peptides from the carboxymethylated L-asparaginase reported in the previous paper, the whole sequence of the enzyme was established. The reported sequence consists of 321 amino acid residues and its calculated molecular weight is 34 080.     

Identification of a potent decatenating enzyme from Escherichia coli

A topoisomerase has been purified from extracts of a topoisomerase I-deficient strain of Escherichia coli based solely on its ability to segregate pBR322 DNA replication intermediates in vitro. This enzyme rapidly decatenated multiply linked form II:form II DNA dimers to form II DNA, provided that the DNA substrate contained single-stranded regions. Efficient relaxation of negatively supercoiled DNA was observed when reaction mixtures were incubated at 52 degrees C, but not at 30 degrees C (the temperature at which decatenation was readily observed). This topoisomerase was insensitive to the DNA gyrase inhibitor norfloxacin and unaffected by antibody directed against topoisomerase I. Relaxation of a unique plasmid topoisomer revealed that this decatenase changed the linking number of the DNA in steps of one and was therefore a type 1 topoisomerase. The cleavage pattern of a fragment of single-stranded phi X174 DNA generated by this decatenase was virtually identical to that reported for topoisomerase III, the least characterized topoisomerase present in E. coli.     

Physiology of L-asparaginase synthesis in recombinants of Escherichia coli A-1.

A mating between Escherichia coli 4318 (thi leu Las- Hfr) and E. coli A-1 (Met- Las+ F-) resulted in the formation of prototrophic recombinants having L-asparaginase activities at three distinct levels. The physiology of L-asparaginase synthesis in these recombinants is decribed. One class of recombinants produced significantly more L-asparaginase than E. coli A-1. L-Asparaginase synthesis in the recombinants was inhibited by the presence of dissolved oxygen in the medium and was transiently repressed by the presence of glucose in the same manner as that observed in the parental strains. L-Asparaginase activity was increased by the addition of oxalacetate as well as other members of the tricarboxylic acid cycle.