The exocellular beta-lactamase of Streptomyces albus G. Purification, properties and comparison with the exocellular DD-carboxypeptidase.

The exocellular beta-lactamase of Streptomyces albus G has been purified to near protein homogeneity. It consists of one single polypeptide chain of mol.wt. 30 000-31 000, has a rather low isoelectric point (at pH 6.0) and contains less lysine (2.1%) and more half-cystine residues than most beta-lactamases from other Gram-positive bacteria. Penicillins are much better substrates than delta 3-cephalosporins; the catalytic-centre activity of good penicillin substrates is 333-500 s-1. The exocellular, mol.wt. 17 000 DD-carboxypeptidase of S. albus G [previously purified to protein homogeneity; Duez, Frère, Geurts, Ghuysen, Dierickx & Delcambe (1978) Biochem. J. 175, 793-800] behaves as an exceedingly poor beta-lactamase, hydrolysing benzylpenicillin into benzylpenicilloate 5 x 10(-6)-fold less rapidly than does the exocellular beta-lactamase. To all appearances, the beta-lactamase has no bivalent cation requirement whereas, as shown elsewhere [Dideberg, Charlier, Dupont, Vermeire, Frère & Ghuysen (1980) FEBS Lett. 117, 212-214, and Dideberg, Joris, Frère, Ghuysen, Weber, Robaye, Delbrouck & Roelands (1980) FEBS Lett. 117, 215-218], the DD-carboxypeptidase possesses one essential Zn2+ ion per molecule. Peptide 'mapping' and immunological studies suggest that the two Streptomyces enzymes probably have very different structural and mechanistic properties.     

The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G. Interaction with beta-lactam antibiotics.

Kinetically, the three-step model proposed for the interaction between beta-lactam antibiotics and the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and Actinomadura R39 [Frère, Ghuysen & Iwatsubo (1975) Eur. J. Biochem. 57, 343--357; Fuad, Frère, Ghuysen, Duez & Iwatsubo (1976) Biochem. J. 155, 623--629] applies to the interaction between the much less penicillin-sensitive exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G and at least phenoxymethylpenicillin, cephalothin and cephalosporin C. The penicillin resistance of the albus G enzyme is mainly due to the low efficiency with which the first reversible complex formed with the antibiotic (complex EI) undergoes transformation into a second more stable complex EI*. Analysis of the ternary interaction between enzyme, NalphaNepsilon-diacetyl-L-lysyl-D-alanyl-D-alanine (Ac2-L-Lys-D-Ala-D-Ala) and cephalosporin C indicates a non-competitive mechanism.     

Interaction of beta-iodopenicillanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39.

The beta-lactamases of Streptomyces albus G and Actinomadura R39 are inactivated by beta-iodopenicillanate. However, in contrast with the beta-lactamase I from Bacillus cereus, they also efficiently catalyse the hydrolysis of the inactivator; with the S. albus G enzyme, kcat. is larger than 25s-1 and the number of turnovers before inactivation is 515. With the A. R39 enzyme, kcat. is larger than 50s-1 and the number of turnovers before inactivation is 80. After hydrolysis of the beta-lactam amide bond, the product rearranges into 2.3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which exhibits an absorption maximum at 305 nm.     

A comparison of DNA cleavage by the restriction enzymes SalPI and PstI.

Methods for obtaining highly active, exonuclease-free, stable preparations of the Streptomyces albus P restriction enzyme SalPI are described. SalPI and its isoschizomer PstI (from the taxonomically distant Providencia stuartii 164) both cleave their recognition sequence (5'-CTGCAG-3') to generate fragments terminating in tetranucleotide 3' extensions whose sequence is 5'-TGCA-3'. Bacteriophage R4G2 DNA, protected against SalPI cleavage by pregrowth on S. albus P, is also protected against PstI cleavage; and total DNA of both S. albus P and P. stuartii 164 is resistant to cleavage by both enzymes.     

Mode of interaction between beta-lactam antibiotics and the exocellular DD-carboxypeptidase--transpeptidase from Streptomyces R39.

The exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R39 is inhibited by beta-lactam antibiotics according to the same general scheme of reaction as the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61. However, the values for the kinetic constants involved in the reaction are very different for the two enzymes and provide an explanation for the observation that the R39 enzyme is more sensitive to beta-lactam antibiotics than the R61 enzyme. Further, particular beta-lactams influence the kinetic constants to different extents depending on the source of the enzyme, so that a physical basis for the spectrum of antibiotic activity against particular enzyme systems is provided.     

Interactions between non-classical beta-lactam compounds and the beta-lactamases of Actinomadura R39 and Streptomyces albus G.

6-Aminopenicillanic acid, 7-aminocephalosporanic acid, mecillinam and quinacillin have varying substrate activities for both the R39 beta-lactamase (excreted by Actinomadura R39) and the G beta-lactamase (excreted by Streptomyces albus G). Cefoxitin and quinacillin sulphone are not recognized by the G beta-lactamase and are weak inactivators of the R39 beta-lactamase. N-Formimidoylthienamycin is a poor substrate for the G beta-lactamase and a potent inactivator of the R39 beta-lactamase. The high value of the bimolecular rate constant for enzyme inactivation is mainly due to a very low dissociation constant (1 microM). Clavulanate is an inactivator of both G and R39 beta-lactamases. The reaction with this latter enzyme is a branched pathway where normal turnover and permanent enzyme inactivation occur concomitantly. Between 28 and 43 molecules of clavulanate are hydrolysed before one of them has the opportunity to inactivate one molecule of enzyme.     

Peptide inhibitors of Streptomyces dd-carboxypeptidases

1. Peptides that inhibit the dd-carboxypeptidases from Streptomyces strains albus G and R61 were synthesized. They are close analogues of the substrates of these enzymes. The enzymes from albus G and R61 strains are in general inhibited by the same peptides, but the enzyme from strain R39 differs considerably. 2. The two C-terminal residues of the peptide substrates and inhibitors appear to be mainly responsible for the initial binding of the substrate to the enzymes from albus G and R61 strains. The side chain in the third residue from the C-terminus seems critical in inducing catalytic activity. 3. Experimental evidence is presented suggesting that the amide bond linking the two C-terminal residues has a cis configuration when bound to the enzymes from strains albus G and R61. 4. The peptide inhibitors are not antibiotics against the same micro-organisms.     

Interaction of clavulanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39.

The reactions of beta-lactamases of Actinomadura R39 and Streptomyces albus G with clavulanate proceed along branched pathways. Both enzymes perform the hydrolysis of this beta-lactam with rather high efficiencies (kcat. = 18s-1 and 52s-1 respectively). If large clavulanate/enzyme ratios are used, complete inactivation of the enzymes is observed. At lower ratios, inactivation is only partial. Irreversible inactivation occurs after 400 and 20000 turnovers for the A. R39 and S. albus G enzymes respectively. With the A. R39 beta-lactamase, a transiently inhibited complex is also formed that remains undetectable with the S. albus G beta-lactamase. Kinetic models are presented and studied for the interaction between clavulanate and both enzymes. A tentative general reaction scheme is also discussed.     

Dexyribonucleic acid polymerases of BHK-21/C13cells. Relationship to the physiological state of the cells, and to synchronous indution of synthesis of deoxyribonuleic acid.

BHK-21/C13 cells were grown in culture under conditions that provided exponentially growing cells and quiescent cells, by modifying the concentration of serum in the growth medium. The high-molecular-weight DNA polymerase (DNA polymerase I) from exponentially growing cells accounted for 90% of the total polymerase activity; the low-molecular-weight DNA polymerase (DNA polymerase II) accounted for the remaining 10%. In quiescent cells, DNA polymerase I contributed only 39% of the total polymerase activity and DNA polymerase II 61%. The total amount of DNA polymerase I in exponentially growing cells was 11.3-fold greater than that in quiescent cells, whereas the amount of DNA polymerase II appeared to be relatively independent of the physiological state of the cells. In an extension of these experiments, cells in a quiescent state (Go cells) were stimulated by the 'serum-step-up' method of Burk (1970) to grow and to enter a synchronous wave of DNA synthesis (S-phase cells), 87% of the cells synthesizing DNA at 20 h after the 'serum-step-up'. During the synchrony experiment, the total cytoplasmic and total nuclear DNA polymerase activities each increased about 4-fold in parallel with the increase in the rate of DNA synthesis. Cytoplasmic polymerase activity was always greater than nuclear polymerase activity. The increases observed were maximal at 20 h after 'serum step-up'. By 26 h, there was a decrease in enzyme activity (8% for cytoplasmic polymerase and 16% for nuclear polymerase, both relative to the maximum at 20 h), but the rate of DNA synthesis had declined by 37% relative to the maximum at 20 h. In Go cells, DNA polymerase II (mol.wt. 46000 +/- 4000) was the predominant species, there being twice as much of it as of the total DNA polymerase I. In these cells there was little DNA polymerase IC and ID; the amounts of IA (mol.wt. 900 times 10(3)-1100 times 10(3)) and IB (mol.wt. 460 times 10(3)-560 times 10(3)) were about equal but small.     

Streptomyces dd-carboxypeptidase as transpeptidases. The specificity for amino compounds acting as carboxyl acceptors

The ability of the water-soluble dd-carboxypeptidases of Streptomyces strains albus G, R61, K11 and R39 to perform transpeptidation was studied. The donor was diacetyl-l-lysyl-d-alanyl-d-alanine, and a whole range of amino acids, peptides and structurally related amino compounds were tested for acceptor function. No compound tested was an acceptor for the enzyme from strain albus G whereas the enzymes from strains R61 and K11 could utilize with varying efficiency a wide range of substances including peptides with N-terminal glycine or d-alanine, omega-amino acids, aminohexuronic acids, 6-aminopenicillanic acid and d-cycloserine. Certain peptides, when present in higher concentration, inhibited the transpeptidation observed at lower concentration. The enzyme from strain R39 would not use any dipeptide as an acceptor, but a few compounds that were not glycine or alpha-amino acids of the d-configuration did function thus. These were d-cycloserine and the lactams of meso- or racemic-diaminoadipic acid.     

Genetic regulation of the cytochrome P-450 system in Drosophila melanogaster. I. Chromosomal determination of some cytochrome P-450-dependent reactions

The genetic regulation of some cytochrome P-450-dependent enzyme activities has been studied in adult Drosophila. Strains having genetically determined high or low enzyme activities were crossed with a marker strain and the metabolism was analyzed in microsomes from hybrids carrying different combinations of chromosomes from the strain under test. High p-nitroanisole (PNA) N-demethylation, biphenyl 3-hydroxylation and an increased amount of a protein with an apparent mol. wt. of 54 000, after SDS-gel electrophoresis of the microsomes in insecticide-resistant Drosophila strains, are inherited as dominant second chromosome traits. A low capacity for benzo[a]pyrene (BP) hydroxylation and 7-ethoxycoumarin O-deethylation in the Hikone R strain is semidominantly inherited in both cases and determined by gene(s) on the third chromosome. A semidominantly inherited high 4-hydroxylation of biphenyl and a high amount of a protein with an apparent mol. wt. of 56 000 in the Oregon R strain are also localized to the second chromosome. The results indicate that several other cytochrome P-450-dependent activities are not regulated by the genes mentioned above. In conclusion, at least three genes regulating the cytochrome P-450 system in Drosophila have been identified.     

Restriction of a bacteriophage of Streptomyces albus G involving endonuclease SalI.

The bacteriophage Pa16, isolated from soil on Streptomyces albus G, was restricted when transferred from an alternative host back to S. albus G. Extracted unmodified Pa16 deoxyribonucleic acid was cleaved at a single site by a cell-free extract of S. albus G. Fractions cleaving Pal6 deoxyribonucleic acid contained the endonuclease SalI first described by J. Arrand, P. Myers, and R. J. Roberts (unpublished data). A mutant of S. albus G was isolated which was defective in both restriction and modification of Pal6. This mutant lacked SalI activity. It is concluded that SalI is the agent of restriction of Pal6 by S. albus G.     

The nature of the microfibrillar glycoproteins of elastic fibres. A biosynthetic study.

1. Cell cultures propagated from foetal bovine ligamentum nuchae synthesized and secreted two glycoproteins, designated MFP I and MFP II, that are closely related to elastic-fibre microfibrils. Glycoproteins MFP I (apparent mol.wt. 150 000) and MFP II (apparent mol.wt. 300 000) were metabolically labelled, separated from other culture-medium components by immunoprecipitation with a specific anti-(microfibrillar protein) serum, and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and sodium dodecyl sulphate/gel-filtration chromatography. 2. Ligament cells also synthesized and secreted fibronectin, but salt-fractionation and immunoprecipitation studies with a specific anti-(cold-insoluble globulin) serum established that neither glycoprotein MFP I nor glycoprotein MFP II was related to fibronectin. 3. The secretion of glycoprotein MFP I, but not that of glycoprotein MFP II, was enhanced by the addition of ascorbate to the culture medium. 4. Ascorbate-supplemented ligament cells incorporated [3H]proline into glycoprotein MFP I, and 36% of the nondiffusible proline residues were hydroxylated, exclusively as 4-hydroxy[3H]proline. Less than 1% of the total proline residues in [3H]proline-labelled glycoprotein MFP II were hydroxylated. 5. Ascorbate-supplemented cells incorporated [14C]lysine into glycoprotein MFP I and 30% of the non-diffusible lysine residues were hydroxylated. 6. Newly secreted glycoprotein MFP I was digested by highly purified bacterial collagenase to yield polypeptide fragments of apparent mol.wts. 50 000 and 30 000. Glycoprotein MFP II was not digested by bacterial collagenase. 7. The results suggest that elastic-fibre microfibrils are composed of a novel collagenous glycoprotein MFP I in association, as yet undefined, with a non-collagenous glycoprotein MFP II.     

ATP-sensitive and ATP-insensitive phosphofructokinase in Escherichia coli K-12.

The purification and kinetic characteristics of two phosphofructokinases are described. Aerobic cultures of Escherichia coli exhibit two types of phosphofructokinase. Both types are dimers of mol. wt 150,000 (subunit mol. wt 73,000), whereas the anaerobic culture of E. coli revealed only one type, which is a tetramer of mol. wt 350,000 (subunit mol. wt 90,000). Type 1 of the aerobic enzyme, representing approximately 70% of the total enzyme activity, is ATP-insensitive, whereas type II and the anaerobic enzyme are ATP-sensitive. The addition of AMP stimulates the tetramer, relieving ATP inhibition, and also the type II dimer, which is, however, inhibited at concentrations higher than 0.5 mM AMP. No effect was observed on the type I dimer of the aerobic preparation. ADP stimulates the tetramer and inhibits type I more strongly than type II of the aerobic dimer. The kinetic characteristics together with the effect of metabolites on these phosphofructokinase types are described and discussed in the light of their importance for the regulatory mechanism of the Pasteur effect.     

Delta 2- and delta 3-cephalosporins, penicillinate and 6-unsubstituted penems. Intrinsic reactivity and interaction with beta-lactamases and D-alanyl-D-alanine-cleaving serine peptidases.

The intrinsic reactivity of delta 2- and delta 3-deacetoxy-7-phenylacetamidocephalosporanates, penicillanate, unsubstituted, 2-methyl- and 2-phenyl-penems and other beta-lactam antibiotics has been expressed in terms of the second-order rate constant (M-1.s-1(OH-)) for the hydrolysis of the beta-lactam amide bond by OH- at 37 degrees C. The values thus obtained have been compared with the second-order rate constants (M-1.s-1(enzyme) for the opening of the same beta-lactam amide bond during interaction with the beta-lactamases of Streptomyces albus G and Actinomadura R39 and the D-alanyl-D-alanine-cleaving serine peptidases of Streptomyces R61 and Actinomadura R39. Depending on the cases, the accelerating effect due to enzyme action and expressed by the ratio M-1.s-1(enzyme)/M-1.s-1(OH) varies from less than 2 to more than 1 x 10(6). The primary parameter that governs enzyme action is the goodness of fit of the beta-lactam molecule to the enzyme cavity rather than its intrinsic reactivity. With the D-alanyl-D-alanine-cleaving serine peptidases, the three penems studied form intermediate complexes characterized by very short half lives of 14-100 s, values significantly lower than those exhibited by most beta-lactam compounds.     

Enzymatic preparation of an immunostimulant, the disaccharide-dipeptide, N-acetyl-beta-D-glucosaminyl-(1----4)-N-acetylmuramyl-L-alanyl-D-is ogl utamine, from a bacterial peptidoglycan

The disaccharide-dipeptide N-acetyl-beta-D-glucosaminyl-(1----4)-N-acetylmuramyl-L-alanyl-D-isog lut amine has been obtained by an enzymatic degradation of the peptidoglycan of Actinomadura R39. The peptidoglycan was hydrolyzed successively by the three following enzymes: lysozyme, DD-carboxypeptidase from Streptomyces albus G and gamma-D-glutamyl-meso-diaminopimelate endopeptidase I from Bacillus sphaericus 9602. The by-products of the last reaction were eliminated by successive ion-exchange and gel-permeation chromatographies. Both chemical analysis and mass spectrometry show that the resulting disaccharide-dipeptide is a pure compound.     

Purification and properties of beta-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro.

Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.     

Des-, syn- and anti-oxyimino-delta 3-cephalosporins. Intrinsic reactivity and reaction with RTEM-2 serine beta-lactamase and D-alanyl-D-alanine-cleaving serine and Zn2+-containing peptidases.

The presence and configuration (syn or anti) of an oxyimino group in the 7 (beta)-acyl side chain of 3-cephems do not modify the intrinsic reactivity of the beta-lactam ring, but have highly enzyme-specific effects. When compared with the corresponding desoxyimino beta-lactam compound: (i) with the plasmid-mediated Escherichia coli RTEM-2 serine beta-lactamase, the substrate activity of the anti isomer is increased and that of the syn isomer is decreased; (ii) with the Streptomyces R61 serine D-alanyl-D-alanine cleaving peptidase (a highly penicillin-sensitive enzyme), the rate of enzyme acylation is not or only little affected when the oxyimino group is in the syn configuration, but is decreased when the oxyimino group is in the anti configuration; (iii) with the Actinomadura R39 serine D-alanyl-D-alanine-cleaving peptidase (an exceedingly highly penicillin-sensitive enzyme), the rate of enzyme acylation is unaffected whatever the configuration of the substituent. The oxidation of the sulphur atom of the dihydrothiazine ring on the beta-face of the molecule makes it both a poorer inactivator of the DD-peptidases and a poorer substrate of the beta-lactamase. The Streptomyces albus G Zn2+-containing D-alanyl-D-alanine-cleaving peptidase (a highly penicillin-resistant enzyme) remains highly resistant to all compounds tested.     

The peptidoglycan crosslinking enzyme system in Streptomyces strains R61, K15 and rimosus

The DD-carboxypeptidase-transpeptidase enzyme system in Streptomyces strain K15 consists of: (1) a membrane-bound transpeptidase capable of performing low DD-carboxypeptidase activity; and (2) a set of DD-carboxypeptidases: (a) membrane-bound, (b) lysozyme-releasable and (c) exocellular, having low transpeptidase activities in aqueous media and at low acceptor concentrations. The DD-carboxypeptidases are related to each other and may belong to the same pathway leading to enzyme excretion. A similar enzyme system occurs in Streptomyces strain R61 except that the membrane-bound DD-carboxypeptidase activity is low when compared with the membrane-bound transpeptidase activity. In Streptomyces rimosus the enzyme system consists almost exclusively of the membrane-bound transpeptidase and the levels of membrane-bound, lysozyme-releasable and exocellular DD-carboxypeptidases are very low.