Proteins of the penicillin biosynthesis pathway.

Two sequential steps are common to the biosynthesis of all penicillin-derived antibiotics: the reaction of three L-amino acids to give L-delta-(alpha-aminoadipoyl)-L-cysteinyl-D-valine, and the oxidation of this tripeptide to give isopenicillin N. Recent studies on the peptide synthetase and oxidase enzymes responsible for these steps have implications for the mechanisms and structures of related enzymes involved in a range of metabolic processes.

Purification and characterization of coenzyme F420-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain delta H.

5,10-Methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain delta H was purified to homogeneity with nearly complete recovery. The aerobically stable monofunctional enzyme catalyzed the reversible oxidation of 5,10-methylene-5,6,7,8-tetrahydromethanopterin to its 5,10-methenyl derivative. For the reaction a midpoint potential E'0 = - 362 mV was calculated at 60 degrees C. The methanogenic electron carrier coenzyme F420 was strictly required as the co-substrate. The dehydrogenase (Mr 216,000) was purified as an apparent hexamer of six identical 36 kDa subunits. Oxidation of 5,10-methylenetetrahydromethanopterin coupled to coenzyme F420 reduction catalyzed by the dehydrogenase with a turnover number of 2400 S-1 proceeded via a ternary complex mechanism. High concentrations of monovalent cations markedly stimulated the reaction.

Comparison of amikacin and gentamicin.

The sensitivity to gentamicin (G) and to amikacin (A) of 25 strains of Escherichia coli (EC), 25 Klebsiella sp. (K), 25 Proteus and Providence sp. (PP), and 25 Ps. aeruginosa (PA) were tested in vitro by the disc method, by the inocula-replicating method, and by a tube dilution technique using 10-5 microorganisms/ml. Bactericidal concentrations active on 50% and 90% of the strains were 1.5 and 4.5 mug/ml for EC, 3 and mug/ml for K, 1.5 and 3 mug/ml for PP, and 3 and 3 mug/ml for PA. Resistance to killing by 12 mug/ml of G was found for 16 strains (1 EC, 9 K, 4 PP, and 2 PA) but only 5 strains were also resistant to 12 mug/ml of A. Conversely, 4 strains (1 EC, 1 PP, and 2 PA) were resistant to 12 mug/ml of A but were killed by 6 mug/ml of G or even less. G (1.5 MG/KG) AND A (5 mg/kg) were administered to 5 bolunteers in a crossover fashion. The mean bactericidal activity of the sera obtained at 1 hr after the injection of G and A was, respectively, 1/4 and 1/4 for ED, 1/4 and 1/8 for K, 1/2 and 1/2 for PP, and 1/2 and 1/2 for PA. Sera obtained in A-treated patients killed at a dilution of 1/8, 8 strains of ED, 17 K, 1 PP, and O PA; at that dilution sera from G-treated patients killed 5 ED, 9 K, O P, and O PA. The main advantage of A over G might be the absence of cross-resistance between A and G for particular pathogens.

Endotracheal antibiotics for the prevention of tracheobronchial infections in tracheotomized unconscious patients. A comparative study of gentamicin and aminosidin-polymyxin B combination.

Endotracheal administration of gentamicin has been compared to the endotracheal administration of aminosidin plus polymyxin B as a preventive measure against tracheobronchial infections in 25 and 22 tracheotomized patients respectively who had been admitted to a neurosurgical unit. Both series were comparable as far as underlying disease, duration of hospitalization, surgical therapy. Both regimens were similarly effective from the bacteriologic and clinical points of view. Both regimens were similarly effective in preventing colonization of bronchial secretions by potential pathogens and were associated with a similar frequency of infectious episodes (eight in each group). The use of aminosidin-polymyxin B combination was associated with a lower incidence of emergence of gentamicin resistant strains, but the endotracheal administration of gentamicin was better tolerated than that of the combination. It is concluded that the combination of aminosidin-polymyxin is a useful alternative to gentamicin for the prevention of bronchopulmonary infections in unconscious tracheotomized patients.

Sisomicin: Bacteriological and clinical evaluation.

A preliminary study was conducted with sisomicin, a new aminoglycoside antibiotic. The drug was administered to 40 patients in doses varying from 1.5 to 3.75 mg/kg/day. Sisomicin proved to be an effective therapy in urinary tract infections and to a lesser extent in wound infections caused by gram-negative rods; favorable results have been observed in 91.6% and in 66.6% of the patients presenting these infections. Toxic reactions involving the hearing function, renal function, and general tolerance were infrequent: they occurred in less than 5% of the patients in this series. (However in approximately 22% of the patients, there was a transient appearance of granular casts in the urine.

Therapy of staphylococcal infections: (a comparative study of cephaloridine and gentamicin).

Two groups of 38 patients have been treated for staphylococcal infection with either cephaloridine (4 gm daily) or gentamicin (320 mg daily) by the intramuscular route. The rate of favorable clinical response was higher among the patients who received cephaloridine (78.8 per cent) than among those who were treated with gentamicin (60.5 per cent). No death related to the infection occurred in the cephaloridine-treated patients. The mean peak and trough antibacterial activity reached in the serum of the patients after injection of the antibiotics was higher in patients receiving cephaloridine (1/64 and 1/16) than in those treated with gentamicin (1/16 and 1/4). Patients who failed to respond to therapy had often a low antibacterial activity of the serum. These studies suggest that the 1/8 level of bactericidal activity should be attained in the serum one hour after the administration of the antibiotics to allow optimal results in staphylococcal infections.

Intestinal colonization with lactobacilli strains in neutropenic patients.

In order to protect granulopenic patients against infections by pathogens from their own digestive tract, large amounts of lactobacilli were given to 5 patients whose intestinal flora had been suppressed by oral antibiotics. In all these cases, lactobacilli failed to prevent spontaneous recolonization of the gastrointestinal tract by enteric bacteria. Six attempts of colonization were made; in spite of the importance of the inoculum administered orally, large numbers of lactobacilli were recovered from stools in only two cases.

[Antimicrobial and surgical treatment of tonsils. Bacteriological and histological data collected on 44 tonsillectomized children]. / Traitement antimicrobien et chirurgical des amygdales. Données bactériologiques et histologiques recueillies chez 44 enfants amygdalectomisés.

The tonsils of 44 operated children were submitted to bacteriological and histological examination. 22 children were given an oral penicillin during 5 days before tonsilectomy. Histological data and bacteriological cultures of pharyngeal exsudate and tonsillar material were very similar in the two groups of treated and non treated patients. These data suggest that, when preventive penicillinic treatment is given, it is worthy to consider the dose, the lasting and the route of administration of the prophylactic antibiotic therapy. Its unreliable activity, and the danger of bacteriemy at the moment of the operation plead for an adequate per-operative antibiotherapy.

Comparative clinical study of doxycycline and minocycline.

Doxycycline and minocycline were given intravenously to patients with a serious underlying disease who presented a pulmonary infection or a wound infection. Both therapies were easy to administer and well tolerated. However they should not be used in the initial therapy since resistant strains may occur. The clinical and bacteriological effectiveness of doxycycline and minocycline were similar. In pneumococcal pulmonary infections, the rates of favorable clinical response to doxycycline and minocycline were 73% and 76% respectively. In infections caused by bacteroides sp.(mainly infections of wounds), doxycycline or minocycline resulted in a 75% rate of favorable clinical responses and in a 71% rate of favorable bacteriological responses. Adverse side effects were rare and of minor importance with the exception of bacteriological colonization by doxycycline-resistant or minocycline-resistant microorganisms. This complica-tion occurred in 39% of the patients who were treated with these drugs and resulted in clinical superinfection in 8% of the patients.

Empiric therapy for cancer patients: comparative study of ticarcillin-tobramycin, ticarcillin-cephalothin, and cephalothin-tobramycin.

Three combinations of antibiotics (cephalothin-tobramycin, cephalothin-ticarcillin, and ticarcillin-tobramycin) were administered empirically to 186 patients with cancer who were suspected of having a life-threatening infection. In approximately one-half of these patients, gram-negative infection was documented bacteriologically and consisted of septicemia in 50% of these patients. The three antimicrobial regimens were similarly effective and resulted in a favorable clinical response in approximately 55% of the patients. The administration of the cephalothin-tobramycin combination was associated with a significantly higher frequency of nephrotoxicity than that of the other two regimens.

Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 microM tungstate contained a benzylviologen-linked aldehyde oxidoreductase. The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000. It contained 0.68 +/- 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit. After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained. Acetaldehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent Km values of 12.5, 10.8, and 20 microM, respectively. The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent Km, 0.55 mM). The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate. In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97. The dithionite-reduced enzyme exhibited a broad signal at low temperature with g = 2.04 and 1.92; this is indicative of a [4Fe-4S]1+ cluster interacting with a second paramagnet, possibly the S = 1 system of W(IV). Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea. The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.

Comparative clinical study of tobramycin and gentamicin.

Gentamicin and tobramycin have been compared in vitro and as single-drug therapy in patients with a serious infection caused by gram-negative rods. In vitro, a slight advantage of tobramycin over gentamicin has been found against Pseudomonas aeruginosa. Cross-resistance between gentamicin and tobramycin has been observed for gentamicin-resistant strains of P. aeruginosa and Providence but was not always present. The clinical effectiveness of gentamicin and tobramycin was similar: 14 (45.1%) out of the 31 patients in each series responded favorably. The clinical results were much better in urinary tract infections (66% of favorable responses) than in wound infections, pulmonary infections, septicemia, and meningitis (26% of favorable responses). The frequency of adverse reactions encountered in the present series was similar for both drugs.

Comparative effectiveness of combinations of amikacin with penicillin G and amikacin with carbenicillin in gram-negative septicemia: double-blind clinical trial.

Preliminary results are presented for an ongoing, double-blind, clinical trial, in which the efficacy of amikacin plus penicillin G (Amik-Pen) and amikacin plus carbenicillin (Amik-Carb) is compared in treatment of severe gram-negative infections superimposed on serious underlying disease. All clinical isolates were sensitive to amikacin in vitro (minimal inhibitory concentration, less than 12 mug/ml). Results in 50 patients with cancer and documented gram-negative infection, 29 of which involved septicemia, were analyzed. In the Amik-Pen group, 40% of 15 cases of septicemia responded favorable to therapy, as compared with 86% of 14 cases of septicemia in the Amik-Carb group; this difference is statistically significant (P less than 0.02). When all patients were considered together, the outcome appeared more favorable (1) in infections caused by pathogens sensitive to both antibiotics used then in those caused by organisms sensitive to amikacin only (83% vs. 43%); (2) when the combined antibiotics demonstrated synergy in virto against the offending pathogen than when the combination was nonsynergistic (83% vs. 38%); and (3) when the peak serum antimicrobial dilution titer was larger than or equal to 1:8 than when titers were lower. The results of this study suggest that routine use of an antibiotic combination that has demonstrable in vitro synergy against the offending pathogen should be considered for the treatment of proven or suspected severe infections due to gram-negative bacilli.

Comparison of sisomicin and gentamicin in bacteriuric patients with underlying diseases of the urinary tract.

Sisomicin and gentamicin (2 mg/kg) were administered in a random fashion to patients with bacteriuria superimposed on abnormalities of the urinary tract. Cure was achieved in a similar number of patients in both groups, but superinfection and reinfection with resistant microorganisms was more frequent in patients receiving gentamicin. Untoward side effects were not frequent in this series, especially if the serious underlying urological disease of most patients is taken into consideration. The susceptibility of the causative pathogens to the antibiotic administered and the severity of the underlying disease were the most important factors in the outcome.

Electron-dense granules in Desulfovibrio gigas do not consist of inorganic triphosphate but of a glucose pentakis(diphosphate).

Under certain growth conditions the sulfate-reducing bacterium Desulfovibrio gigas forms electron-dense granules in the cells which had been claimed to consist of a magnesium triphosphate). We observed granules after cultivation in media with a low Fe2+ or NH4+ concentration and reinvestigated the nature of the electron-dense bodies. Energy-dispersive X-ray analysis of the granules in the cells showed that they contain large amounts of P, Mg, and K. Gel electrophoresis and chromatographic analyses of isolated granules which had been dissolved in 20 mM EDTA, however, revealed discrepancies with commercially available polyphosphates. 31P-NMR spectra also lacked the peaks in the -22-ppm region which are characteristic for inner phosphates of polyphosphates confirming that the phosphocompound as isolated from the electron-dense bodies of D. gigas did not consist of polyphosphates. Using multinuclear NMR spectroscopy we showed that the electron-dense bodies of D. gigas contained a novel metabolite which was identified as alpha-glucose 1,2,3,4,6-pentakis(diphosphate).

Purification and properties of 5,10-methylenetetrahydromethanopterin reductase, a coenzyme F420-dependent enzyme, from Methanobacterium thermoautotrophicum strain delta H.

5,10-Methylenetetrahydromethanopterin reductase was purified 22-fold to apparent homogeneity from the methanogenic bacterium Methanobacterium thermoautotrophicum. The enzyme catalyzes the reduction of 5,10-methylene- to 5-methyltetrahydromethanopterin. The electron carrier coenzyme F420 is specifically used as the cosubstrate. The reductase reaction may proceed in both directions, methylene reduction is, however, thermodynamically favored. In addition, the velocity of the reaction in this direction exceeds the reverse reaction by a factor of 26. The reductase is composed of a single subunit with an estimated Mr = 35,000. The active enzyme does not contain a flavin prosthetic group or iron-sulfur clusters, in contrast to 5,10-methylenetetrahydrofolate reductases purified from eukaryotic and eubacterial sources, which catalyze an analogous reaction as the methanogenic reductase.

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas.

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH.

Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies.

The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.

Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation.

The biosynthesis of penicillin and cephalosporin antibiotics in microorganisms requires the formation of the bicyclic nucleus of penicillin. Isopenicillin N synthase (IPNS), a non-haem iron-dependent oxidase, catalyses the reaction of a tripeptide, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV), and dioxygen to form isopenicillin N and two water molecules. Mechanistic studies suggest the reaction is initiated by ligation of the substrate thiolate to the iron centre, and proceeds through an enzyme-bound monocyclic intermediate. Here we report the crystal structure of IPNS complexed to ferrous iron and ACV, determined to 1.3 A resolution. Based on the structure, we propose a mechanism for penicillin formation that involves ligation of ACV to the iron centre, creating a vacant iron coordination site into which dioxygen can bind. Subsequently, iron-dioxygen and iron-oxo species remove the requisite hydrogens from ACV without the direct assistance of protein residues. The crystal structure of the complex with the dioxygen analogue, NO and ACV bound to the active-site iron supports this hypothesis.