Glycine fermentation by Clostridium histolyticum

Clostridium histolyticum grew on glycine, arginine, or threonine as sole substrate. Arginine degradation preceded that of glycine and partially inhibited that of threonine when two amino acids were present. Each amino acid seemed to be individually catabolized, not by a Stickland type of reaction. Glycine fermentation required the presence of complex ingredients. Therefore, an effect of selenite on glycine catabolism could only be demonstrated after scavenging selenium contamination by preculturing Peptostreptococcus glycinophilus in that medium. C. acidiurici was not suited as selenium accumulating organism as C. histolyticum was inhibited by the residual uric acid. Arginine catabolism was unaffected by seleniuum depriviation. The labelling pattern obtained in acetate after incubation of C. histolyticum with [1-¹⁴C]- or [2-¹⁴C]glycine strongly indicated the metabolism of glycine via the glycine reductase pathway.     

A novel aminopeptidase from Clostridium histolyticum

An aminopeptidase was found in the culture filtrate of Cl. histolyticum and purified to homogeneity (130 times) in a two-step procedure. All types of N-terminal amino acids, including proline and hydroxyproline are cleaved by the enzyme from small peptides and from polypeptides. A low rate of hydrolysis was observed for β-naphthylamides and for alanine amide; p-nitroanilides were not hydrolyzed. Kinetic parameters (K_m and V_max) for several tripeptides and the tetrapeptide Pro-Gly-Pro-Pro were determined. The enzyme has a pH optimum at 8.6. The presence of either Mn⁺⁺ or Co⁺⁺ is essential for its activity. Only slight activation was observed with Ni⁺⁺ and Cd⁺⁺, while Zn⁺⁺ and Cu⁺⁺ were inhibitory. The molecular weight of the native enzyme is about 340,000, and a molecular weight of about 60,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate (SDS).The culture filtrate of Cl. histolyticum has been shown to contain various proteolytic enzymes, in addition to collagenase^1–5. In a search for enzymes acting on proline-rich peptides, we tested the crude filtrate with (Pro-Gly-Pro)_n, (Pro-Gly-Pro)_n-OMe, α,DNP-(Pro-Gly-Pro)_n and poly-L-proline as substrates. Proline was formed only from (Pro-Gly-Pro)_n and its methyl ester. This showed the presence in Cl. histolyticum filtrate of an aminopeptidase which cleaves N-terminal proline from polypeptides but not from polyproline. The purification and some of the properties of this clostridial aminopeptidase (CAP) are described in this communication.     

Substrate specificity of beta-collagenase from Clostridium histolyticum

The substrate specificity of beta-collagenase from Clostridium histolyticum has been investigated by measuring the rate of hydrolysis of more than 50 tri-, tetra-, penta-, and hexapeptides covering the P3 to P3' subsites of the substrate. The choice of peptides was patterned after sequences found in the alpha 1 and alpha 2 chains of type I collagen. Each peptide contained either a 2-furanacryloyl (FA) or cinnamoyl (CN) group in subsite P2 or the 4-nitrophenylalanine (Nph) residue in subsite P1. Hydrolysis of the P1-P1' bond produces an absorbance change in these chromophoric peptides that has been used to quantitate the rates of their hydrolysis under first order conditions ([S] much less than KM) from kcat/KM values have been obtained. The identity of the amino acids in all six subsites (P3-P3') markedly influences the hydrolysis rates. In general, the best substrates have Gly in subsites P3 and P1', Pro or Ala in subsite P2', and Hyp, Arg, or Ala in subsite P3'. This corresponds well with the frequency of occurrence of these residues in the Gly-X-Y triplets of collagen. In contrast, the most rapidly hydrolyzed substrates do not have residues from collagen-like sequences in subsites P2 and P1. For example, CN-Nph-Gly-Pro-Ala is the best known substrate for beta-collagenase with a kcat/KM value of 4.4 X 10(7) M-1 min-1, in spite of the fact that there is neither Pro nor Ala in P2 or Hyp nor Ala in P1. These results indicate that the previously established rules for the substrate specificity of the enzyme require modification.     

An extracellular aminopeptidase from Clostridium histolyticum.

An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated. From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme. The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin. Low rates of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X. The highest Kcat values are observed with proline and alanine. In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues. The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+. The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively. The optimal temperature is 40 degrees C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate.     

Agglutination,Toxigenicity and Sorbitol Fermentation of Clostridium difficile

A total of 79 Clostridium difficile strains from different sources (50 strains from the fecal specimens of healthy adults, 13 from patients receiving antibiotics without gastrointestinal complications, 13 from antibiotic-associated pseudomembranous colitis (PMC) or diarrhea patients, and three strains from ATCC) were investigated for agglutinability, using formol-treated cells as antigen, in relation to toxigenicity. C. difficile strains tested were divided into four serovars, I, II, III, and IV, by the cross-agglutination test. The agglutinin absorption test revealed that strains of serovar I, agglutinable with high titers (5,120–10,240) to antiserum prepared against a highly toxigenic C. difficile strain, ATCC 17859, possessed the serovar-specific antigen. All of the strains of serovar I were highly toxigenic and all 13 strains isolated from the fecal specimens of antibiotic-associated PMC or diarrhea patients belonged to this serovar, whereas 19 (38%) out of 50 strains from healthy adults and four (30.8%) out of 13 strains from patients receiving antibiotics without gastrointestinal complications possessed this antigen. None of the strains of other clostridial species than C. difficile were agglutinated by the three reference antisera used. Further study on the sugar fermentation test disclosed that the sorbitol-fermenting property of C. difficile is very closely related to the toxigenicity and agglutinability.