Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Novel properties of Escherichia coli exonuclease III.

The specificity of hydrolysis of polynucleotide termini by Escherichia coli exonuclease III was studied with the use of oligothymidylate annealed to polydeoxyadenylate. The size of the products after 3' leads to 5'-hydrolysis of 5'-labeled substrate is temperature-dependent. At 25 degrees the enzyme can hydrolyze a polynucleotide chain up to the last 5'-terminal dinucleotide. A gradation of higher 5'-terminal oligonucleotides of defined chain lengths is produced after limit digestion by the enzyme when the temperature is raised between 25 degrees to 60 degrees. When the oligothymidylate was labeled at the 3'-ends with ribonucleotides, it was observed that exonuclease III can cleave a single or two consecutive ribonucleotides regardless of whether the ribonucleotides are base-paired or mismatched.     

Complete nucleotide sequence of the Escherichia coli ptr gene encoding protease III.

The nucleotide sequence of a 3120 bp region of the E. coli chromosome that includes the entire ptr gene has been determined. The proposed coding region for Protease III is 2889 nucleotides long, which would encode a protein consisting of 962 amino acids with a calculated molecular mass of 107,719 daltons. The predicted primary structure of the protein includes a 23-residue signal sequence, cleavage of which would give rise to a mature protein of molecular mass 105,124 daltons. At its 3' end, the ptr gene overlaps the start of the recB coding sequence by 8 bases, suggesting that these genes may form part of an operon.     

Specificity and enzymatic mechanism of the editing exonuclease of Escherichia coli DNA polymerase III.

Exonucleolytic editing is a major contributor to the fidelity of DNA replication by the multisubunit DNA polymerase (pol) III holoenzyme. To investigate the source of editing specificity, we have studied the isolated exonuclease subunit, epsilon, and the pol III core subassembly, which carries the epsilon, theta, and alpha (polymerase) subunits. Using oligonucleotides with specific terminal mismatches, we have found that both epsilon and pol III core preferentially excise a mispaired 3' terminus and therefore have intrinsic editing specificity. For both epsilon and pol III core, exonuclease activity is much more effective with single-strand DNA; with a double-strand DNA, the exonuclease is strongly temperature-dependent. We conclude that the epsilon subunit of pol III holoenzyme is itself a specific editing exonuclease and that the source of specificity is the greater melting capacity of a mispaired 3' terminus.     

Physical characterization of the cloned protease III gene from Escherichia coli K-12.

Analysis of the cloned protease III gene (ptr) from Escherichia coli K-12 has demonstrated that in addition to the previously characterized 110,000-Mr protease III protein, a second 50,000-Mr polypeptide (p50) is derived from the amino-terminal end of the coding sequence. The p50 polypeptide is found predominantly in the periplasmic space along with protease III, but does not proteolytically degrade insulin, a substrate for protease III. p50 does not appear to originate from autolysis of the larger protein. Protease III is not essential for normal cell growth since deletion of the structural gene causes no observed alterations in the phenotypic properties of the bacteria. A 30-fold overproduction of protease III does not affect cell viability. A simple new purification method for protease III is described.     

Escherichia coli photoreactivating enzyme: purification and properties

We have purified large quantities of Escherichia coli photoreactivating enzyme (EC 4.1.99.3) to apparent homogeneity and have studied its physical and chemical properties. The enzyme has a molecular weight of 36 800 and a S020,W of 3.72 S. Amino acid analysis revealed an apparent absence of tryptophan, a low content of aromatic residues, and the presence of no unusual amino acids. The N terminus is arginine. The purified enzyme contained up to 13% carbohydrate by weight. The carbohydrate was composed of mannose, galactose, glucose, and N-acetylglucosamine. The enzyme is also associated with RNA (approximately 10 nucleotides/enzyme molecule) containing uracil, adenine, guanine, and cytosine with no unusual bases detected.     

Beta-glycosidase of Thermus thermophilus KNOUC202: gene and biochemical properties of the enzyme expressed in Escherichia coli

The beta-glycosidase gene of Thermus thermophilus KNOUC202 was cloned, expressed in Escherichia coli JM109(DE3), and the enzyme was purified and characterized. The gene (KNOUC202/beta-gly) was composed of 1296 bp encoding a beta-glycosidase (KNOUC202beta-glycosidase) of 431 a.a., belonging to the family 1 of glycosyl hydrolase. The gene was expressed as monomer of 430 a.a. with amino terminal methionine excised in E. coli JM109(DE3). The enzyme hydrolyzed beta-glycosides whose glycone are galactose, glucose and fucose well, however showed no or very low activity on beta-D-glycosides whose glycone are disaccharides and xylose. kcat of the enzyme for the hydrolysis of p-Nph-beta-D-Glcp was lower than those for p-Nph-beta-D-Galp and ONPG, however K(m) for p-Nph-beta-D-Glcp was highly lower than those for p-Nph-beta-D-Galp and ONPG resulting in the catalytic efficiency(k(cat)/K(m)) for the hydrolysis of p-Nph-beta-D-Glcp much higher than those for p-Nph-beta-D-Galp and ONPG. Optimum pH and optimum temperature of the enzyme were pH 5.4 and 90 degrees C. The enzyme has high thermostability, not losing its activity at 80 degrees C for 2 h in 0.05 M Na-phosphate buffer of pH 6.8 with T(m) of 100.0 +/- 0.031 degrees C in 0.02 M Tris-HCl buffer of pH 8.2. The beta-glycosidase produced a disaccharide composed of galactose as transglycosylation byproduct during hydrolysis of lactose.     

Cytidine deaminase from Escherichia coli B. Purification and enzymatic and molecular properties

Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) from Escherichia coli has been purified to homogeneity through a rapid and efficient two-step procedure consisting of anion-exchange chromatography followed by preparative electrophoresis. The final preparation is homogeneous, as judged by a single band obtained by disc gel electrophoresis performed in the absence and presence of denaturing agents. The native protein molecular weight determined by gel filtration is 56 000. Sodium dodecyl sulfate disc gel electrophoresis experiments conducted upon previous incubation of the enzyme with dimethyl suberimidate suggest an oligomeric structure of two identical subunits of 33 000 molecular weight. The absorption spectrum of the protein reveals a maximum at 277 nm and a minimum at 255 nm. The isoelectric point is at pH 4.35. Amino acid analysis indicates an excess of acidic amino acid residues as well as six half-cystine residues. No interchain disulfide groups have been evidenced. According to Cleland's nomenclature, kinetic analysis shows a rapid-equilibrium random Uni-Bi mechanism. Cytidine deaminase is competitively inhibited by various nucleosides. Km values for cytidine, deoxycytidine, and 5-methylcytidine are 1.8 X 10(-4), 0.9 X 10(-4), and 12.5 X 10(-4) M, respectively.     

Crystallization and crystallographic characterization of the iron-sulfur-containing DNA-repair enzyme endonuclease III from Escherichia coli.

Endonuclease III from Escherichia coli is an iron-sulfur enzyme possessing both DNA N-glycosylase and apurinic/apyrimidinic lyase activities. It could serve to repair damaged thymine residues in DNA via base excision-repair. We have crystallized endonuclease III by a combination of dialysis and seeding techniques after exploration of a wide variety of precipitants which failed to yield macroscopic crystals. Important features of the optimized crystallization include: the use of 5 to 10% glycerol, a temperature of 15 degrees C, controlled dialysis to decrease ionic strength and macroseeding using a 200 mM-NaCl transfer buffer to dissolve microcrystalline contamination. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 48.5 A, b = 65.8 A, c = 86.8 A, alpha = beta = gamma = 90 degrees, have one 23 kDa monomer per asymmetric unit, and diffract to 1.84 A. A native anomalous Patterson map located the iron-sulfur cluster and reaffirmed its existence. The reported crystallization procedures ensure an ample supply of crystals for the extensive heavy-atom derivative search necessary for this labile iron-sulfur enzyme. The elucidation of endonuclease III structure will facilitate not only the understanding of glycosylase and lyase mechanisms but also the structure and function of this new class of iron-sulfur proteins.     

The physical and enzymatic properties of Escherichia coli recA protein display anion-specific inhibition.

The enzymatic activities of Escherichia coli recA protein are sensitive to ionic composition. Here we report that sodium glutamate (NaGlu) is much less inhibitory to the DNA strand exchange, DNA-dependent ATPase, and DNA binding activities of the recA protein than is NaCl. Both joint molecule formation and complete exchange of DNA strands occur (albeit at reduced rates) at NaGlu concentrations as high as 0.5 M whereas concentrations of NaCl greater than 0.2 M are sufficient for complete inhibition. The single-stranded DNA (ssDNA)-dependent ATPase activity is even less sensitive to inhibition by NaGlu; ATP hydrolysis stimulated by M13 ssDNA is unaffected by 0.5 M NaGlu and is further stimulated by E. coli ssDNA binding protein approximately 2-fold. Finally, NaGlu has essentially no effect on the stability of recA protein-epsilon M13 DNA complexes, with concentrations of NaGlu as high as 1.5 M failing to dissociate the complexes. Surprisingly, NaGlu also has little effect on the concentration of NaCl required to disrupt the recA protein-epsilon M13 DNA complex, demonstrating that destabilization is dependent on both the concentration and type of anionic rather than cationic species. Quantitative analysis of DNA binding isotherms establishes that the intrinsic binding affinity of recA protein is affected by the anionic species present and that the cooperativity parameter is relatively unaffected. Consequently, the sensitivity of recA protein-ssDNA complexes to disruption by NaCl does not result from the competitive effects associated with cation displacement from the ssDNA upon protein binding but rather results from anion displacement upon complex formation. The magnitude of this anion-specific effect on ssDNA binding is large relative to that of other nucleic acid binding proteins.     

Regulation of gluconeogenesis by the glucitol enzyme III of the phosphotransferase system in Escherichia coli.

The gut operon was subcloned into various plasmid vectors (M. Yamada and M. H. Saier, Jr., J. Bacteriol. 169:2990-2994, 1987). Constitutive expression of the plasmid-encoded operon prevented utilization of alanine and Krebs cycle intermediates when they were provided as sole sources of carbon for growth. Expression of the gutB gene alone (encoding the glucitol enzyme III), subcloned downstream from either the lactose promoter or the tetracycline resistance promoter, inhibited utilization of the same compounds. On the other hand, overexpression of the gutA gene (encoding the glucitol enzyme II) inhibited the utilization of a variety of sugars as well as alanine and Krebs cycle intermediates by an apparently distinct mechanism. Phosphoenolpyruvate carboxykinase activity was greatly reduced in cells expressing high levels of the cloned gutB gene but was nearly normal in cells expressing high levels of the gutA gene. A chromosomal mutation in the gutR gene, which gave rise to constitutive expression of the chromosomal gut operon, also gave rise to growth inhibition on gluconeogenic substrates as well as reduced phosphoenolpyruvate carboxykinase activity. Phosphoenolpyruvate synthase activity in general varied in parallel with that of phosphoenolpyruvate carboxykinase. These results suggest that high-level expression of the glucitol enzyme III of the phosphotransferase system can negatively regulate gluconeogenesis by repression or inhibition of the two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase.     

Characterization of an intracellular insulin-degrading enzyme in human erythrocytes

Using conventional techniques of ammonium sulfate fractionation and Sephadex gel column chromatography, insulin-degrading enzyme was partially purified from lysate of human erythrocytes. The enzymatic activity was measured by the trichloroacetic acid precipitation method. Compared to trypsin, the enzyme was highly specific for insulin. The apparent molecular weight of the enzyme was 160,000 Da, the optimum pH was the 7.4 to 7.8 range, and the Km value for insulin for the partially purified enzyme was 162 nM. Bacitracin and N-ethylmaleimide were potent inhibitors, while chloroquine, ethylenediaminetetraacetate, antipain, and soybean trypsin inhibitor failed to inhibit the activity of the enzyme. Like most nucleated cells, human erythrocytes not only have the membranal insulin receptors, but also possess the cytosolic specific insulin-degrading enzyme. Insulin internalization and degradation are shown to be due to the receptor and the enzyme acting in concert as in many nucleated cells. Anucleated erythrocytes have both these entities for possible internalization and degradation of insulin.     

Assembly and enzymatic properties of the catalytic domain of human complement protease C1r.

The catalytic properties of C1r, the protease that mediates activation of the C1 complex of complement, are mediated by its C-terminal region, comprising two complement control protein (CCP) modules followed by a serine protease (SP) domain. Baculovirus-mediated expression was used to produce fragments containing the SP domain and either 2 CCP modules (CCP1/2-SP) or only the second CCP module (CCP2-SP). In each case, the wild-type species and two mutants stabilized in the proenzyme form by mutations at the cleavage site (R446Q) or at the active site serine residue (S637A), were produced. Both wild-type fragments were recovered as two-chain, activated proteases, whereas all mutants retained a single-chain, proenzyme structure, providing the first experimental evidence that C1r activation is an autolytic process. As shown by sedimentation velocity analysis, all CCP1/2-SP fragments were dimers (5.5-5.6 S), and all CCP2-SP fragments were monomers (3.2-3.4 S). Thus, CCP1 is essential to the assembly of the dimer, but formation of a stable dimer is not a prerequisite for self-activation. Activation of the R446Q mutants could be achieved by extrinsic cleavage by thermolysin, which cleaved the CCP2-SP species more efficiently than the CCP1/2-SP species and yielded enzymes with C1s-cleaving activities similar to their active wild-type counterparts. C1r and its activated fragments all cleaved C1s, with relative efficiencies in the order C1r < CCP1/2-SP < CCP2-SP, indicating that CCP1 is not involved in C1s recognition.     

Saturation and specificity of the Lon protease of Escherichia coli.

Lon is an ATP-dependent protease of Escherichia coli. The lon mutation has a pleiotropic phenotype: UV sensitivity, mucoidy, deficiency for lysogenization by bacteriophage lambda and P1, and lower efficiency in the degradation of abnormal proteins. All of these phenotypes are correlated with the loss of protease activity. Here we examine the effects of overproduction of one Lon substrate, SulA, and show that it protects two other substrates from degradation. To better understand this protection, we mutagenized the sulA gene and selected for mutants that have partially or totally lost their ability to saturate the Lon protease and thus can no longer protect another substrate. Some of the SulA mutants lost their ability to protect RcsA from degradation but could still protect the O thermosensitive mutant protein (Ots). All of the mutants retained their capacity to induce cell division inhibition. It was also found that deletion of the C-terminal end of SulA affected its activity but did not affect its susceptibility to Lon. We propose that Lon may have more than one specificity for peptide cleavage.