Intracellular GSH of Streptococcus thermophilus shows anti-oxidative activity against low-density lipoprotein oxidation in vitro and in a hyperlipidaemic hamster model.

Streptococcus thermophilus YIT 2001 (ST-1), a lactic acid bacterial strain, was shown to have inhibitory effects on the oxidation of low-density lipoprotein (LDL) and the development of aortic fatty lesions in an animal model, and lower the serum levels of malondialdehyde-modified LDL, an oxidative modification product of LDL, in a clinical trial. This study aimed to identify the intracellular active component of ST-1 associated with anti-oxidative activity against LDL oxidation. High-performance liquid chromatography-electrospray ionisation mass spectrometry analysis after fractionation of the cellular extract by reversed-phase chromatography demonstrated that the active fraction contained reduced glutathione (GSH). GSH showed anti-oxidative activity in a dose-dependent manner, while this activity disappeared following thiol derivatisation. ST-1 had the strongest anti-oxidative activity against LDL oxidation and the highest level of intracellular GSH among five strains of S. thermophilus. In addition, the anti-oxidative activity of ST-1 after thiol derivatisation decreased by about half, which was similar to that of three other strains containing poor or no intracellular GSH or thiol components. Moreover, anti-oxidative activity against LDL oxidation was observed in hyperlipidaemic hamsters fed with high GSH ST-1 cells but not in those given low GSH cells. These findings suggest that intracellular GSH in ST-1 may provide beneficial effects via anti-oxidative activity against LDL oxidation and excess oxidative stress in the blood.

The role of Pro-239 in the catalysis and heat stability of subtilisin E.

Site-directed mutagenesis was employed to analyze the role of an alpha-helix containing catalytic Ser-221 of subtilisin E. Pro-239 located at the carboxy-terminal end of the alpha-helix was first replaced with Gly to examine the role of Pro-239 in the catalysis and stability of subtilisin E. The mutation was found to decrease both the catalytic rate (kcat) and the heat stability. This result strongly suggests that Pro-239 plays an important role in the maintenance of the alpha-helix, affecting the functioning of the active site. Various amino acid substitutions at position 239 were attempted to obtain the active subtilisins from Gly-239 subtilisin. Lys- and Arg-substitutions were found to result in more active and stable subtilisins than the Gly-239 subtilisin. In particular, the Arg-239 mutant showed enhanced heat stability compared with the wild type. These results demonstrate the important role of the alpha-helix containing catalytic Ser-221 in the catalysis as well as in the heat stability of subtilisin.

Molecular characterization of a cell wall-associated proteinase gene from Streptococcus lactis NCDO763.

Streptococcus lactis NCDO763 harbours a plasmid designated pLP763. The cells harbouring pLP763 are able to grow to a higher density in milk because of their proteinase-positive phenotype (Prt+). The 6.2 kb HindIII-PstI fragment from pLP763 was found to be responsible for the Prt+ phenotype. The DNA fragment contains an incomplete large open reading frame (ORF). Further sequence analysis downstream from the PstI site revealed that the ORF consists of 5706 bases. It was found that the deduced amino acid sequence consisting of 1902 amino acid residues was extremely similar to that of the Wg2 proteinase, a serine protease from Streptococcus cremoris, suggesting that both genes were derived from a common ancestral gene.

Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis.

The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme. Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis. Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space. Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme. The other six mutant enzymes showed a marked decrease in activity. This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure. The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature. It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates.

In vitro processing of pro-subtilisin produced in Escherichia coli.

In a previous paper (Ikemura, H., Takagi, H., and Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864), we demonstrated that the pro-sequence consisting of 77 amino acid residues at the amino terminus of subtilisin is essential for the production of active subtilisin. When the aggregates of pro-subtilisin produced in Escherichia coli were solubilized in 6 M guanidine hydrochloride and dialyzed against 200 mM sodium phosphate buffer (pH 7.1 or 6.2), pro-subtilisin was efficiently processed to active subtilisin. When more than 14 residues were removed from the amino terminus of the pro-sequence, active subtilisin was no longer produced as in the in vivo experiments. Similarly, active subtilisin would not renature under the same conditions once solubilized in guanidine hydrochloride. When the aspartic acid residue at the active site (Asp32) was altered to asparagine, processing of mutant pro-subtilisin was not observed even in the presence of wild-type pro-subtilisin. Inhibitors such as phenylmethanesulfonyl fluoride or Streptomyces subtilisin inhibitor did not block the processing of wild-type pro-subtilisin. These facts indicate that processing or pro-subtilisin is carried out by an intramolecular, self-processing mechanism. When the sample was dialyzed against 20 mM sodium phosphate (pH 6.2), no active subtilisin was found, suggesting that the highly charged nature of the pro-sequence plays an important role in the process of refolding of denatured pro-subtilisin.

Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli.

Subtilisin E, an alkaline serine protease of Bacillus subtilis 168, is first produced as a precursor, pre-pro-subtilisin, which consists of a signal peptide for protein secretion (pre-sequence) and a peptide extension of 77 amino acid residues (pro-sequence) between the signal peptide and mature subtilisin. When the entire coding region for pre-pro-subtilisin E was cloned into an Escherichia coli expression vector, active mature subtilisin E was secreted into the periplasmic space. When the pre-sequence was replaced with the E. coli OmpA signal peptide, active subtilisin E was also produced. When the OmpA signal peptide was directly fused to the mature subtilisin sequence, no protease activity was detected, although this product had the identical primary structure as subtilisin E as a result of cleavage of the OmpA signal peptide and was produced at a level of approximately 10% of total cellular protein. When the OmpA signal peptide was fused to the 15th or 44th amino acid residue from the amino terminus of the pro-sequence, active subtilisin was also not produced. These results indicate that the pro-sequence of pre-pro-subtilisin plays an important role in the formation of enzymatically active subtilisin. It is proposed that the pro-sequence is essential for guiding appropriate folding of the enzymatically active conformation of subtilisin E.

Essential structure for full enterotoxigenic activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli.

Several analogues of heat-stable enterotoxins (STh and STp) produced by enterotoxigenic Escherichia coli were synthesized. Peptides (STh[6-18] and STp[5-17]) consisting of 13 amino acid residues from the Cys residue near the N-terminus to the Cys residue near the C-terminus and linked by three disulfide bonds had the same biological and immunological properties as native STh and STp, respectively. The results indicated that the sequence with the 13 amino acid residues and three disulfide linkages is essential for full biological activity of ST.

Chemical synthesis of a highly potent and heat-stable analog of an enterotoxin produced by a human strain of enterotoxigenic Escherichia coli.

A shorter analog of a heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli SK-1, consisting of 14 amino acid residues including 6 half-cystine residues, was synthesized by conventional methods. The peptide was evaluated for ability to induce intestinal secretion in suckling mice and for stability at high temperature under various conditions. The peptide was 2-5 times more potent than native toxin and was still toxic after heat-treatment at 120 degrees C for 30 min.