Biosynthesis of 2-deoxystreptamine.

Extracts of Streptomyces fradiae 75-078, a producer of an antibiotic neomycin, were found to catalyze three reactions which are included in the proposed biosynthetic pathway to 2-deoxystreptamine; amination of deoxyinosose yielding deoxyinosamine, conversion of deoxyinosamine to 2-deoxystreptamine and deamination of 2-deoxystreptamine. Glutamine was effective as an amino-donor for both transamination reactions; conversions of deoxyinosose to deoxyinosamine and of aminodeoxyinosose to 2-deoxystreptamine. Conversion of deoxyinosamine to 2-deoxystreptamine, presumably including successive dehydrogenation and transamination at position 1, was stimulated by NAD+. On DEAE-Sepharose CL-6B ion-exchange chromatography, the enzyme activity catalyzing amination of deoxyinosose and deamination of 2-deoxystreptamine was eluted as an entity (aminotransferase), while the one converting deoxyinosamine to 2-deoxystreptamine, only if the aminotransferase is supplemented, can be eluted as a separate peak (deoxyinosamine dehydrogenase). The molecular weight of the aminotransferase was estimated to be 130,000 daltons by chromatography on Sepharose CL-6B. Enzymatic synthesis of 2-deoxystreptamine from deoxyinosose was demonstrated by the cell free extracts.

Purification and properties of an enzyme reducing leupeptin acid to leupeptin.

An enzyme catalyzing the reduction of leupeptin acid to leupeptin was partially purified from a cell extract of Streptomyces roseus MA839-A1, a leupeptin producer. The enzyme was tentatively named leupeptin acid reductase. The molecular weight was estimated to be 320,000 by chromatography on Sepharose 6B. The reductase eluted with leupeptin acid synthetase both in molecular sieve chromatography and in affinity chromatography. The main properties of the reductase were: (1) ATP and NADPH were required for activity. ATP could not be replaced by GTP, ADP or AMP. NADPH could not be replaced by NADH. (2) Michaelis constants for ATP and NaDPH were 4.2 . 10(-5) M and 1.3 10(-6) M, respectively. (3) The enzyme was inhibited by leupeptin, the reaction product, and antipain. Both inhibitors have an L-argininal residue at the C-terminal structure. (4) The enzyme did not catalyze the conversion of leupeptin to leupeptin acid. Leupeptin acid reductase and leupeptin acid synthetase were found in the 10,000 x g pellet of the cell homogenate. The reductase was not released as readily from the pellet as the synthetase either by washing or by repeated freeze-thawing. Synthesis of leupeptin from acetyl-CoA, L-lucine and L-arginine in vitro was accomplished by combining leucine acyltransferase and the enzyme complex consisting of leupeptin acid synthetase an leupeptin acid reductase.

Studies on the effects of 3-acetyl-4"-isovaleryltylosin against multiple-drug resistant strains of Staphylococcus aureus.

The macrolide-resistance of multiple-drug resistant strains of Staphylococcus aureus was divided into two types; the decreased sensitivity of ribosomes (type I) and the decreased uptake (type II). Both types were resistant to erythromycin, tylosin and 3-acetyltylosin, and their resistance was not inducible. 3-Acetyl-4"-isovaleryltylosin inhibited the growth of both types. Protein synthesis on ribosomes of type I in vitro (S. aures MS-9610) was inhibited by 3-acetyl-4"-isovaleryltylosin, but little or no inhibition was seen with either tylosin or 3-acetyltylosin. Ribosomes of type II in vitro (S. aureus MS-8710) were sensitive to all macrolides. 3-Acetyl-4"-isovaleryltylosin accumulated about twice as much as 3-acetyltylosin in intact cell of type II.

Biosynthesis of leupeptin. IV. Is protein turnover in leupeptin producer cells affected by leupeptin?

There was no significant difference between the rates of protein degradation in cells of a leupeptin-producing strain and that of a leupeptin-nonproducing strain, the latter being derived from the former by mutation. Protein autodigestion in a cell homogenate of the leupeptin producer was sensitive to EDTA and chymotrypsin and less sensitive to leupeptin. On the contrary, protein degradation caused by exogenous trypsin in a similar homogenate was highly sensitive to leupeptin. A labeling experiment with [14C]-arginine of a culture of the leupeptin producer strain revealed that leupeptin was accumulated mostly in the medium and only slightly in the cells; the ratio between the amount in the medium and that in the cells was about 250:1. In contrast, leupeptin acid, the proximal intermediate having no antiplasmin activity, showed a ratio of 5:1.

Biosynthesis of leupeptin. III. Isolation and properties of an enzyme synthesizing acetyl-L-leucine.

An enzyme which catalyzes acetylation of L-leucine with acetyl-CoA was partially purified from a cell extract of Streptomyces roseus MA839-A1, a leupeptin producer. The molecular weight of this enzyme is about 27,000 daltons. The enzyme has fairly broad specificity for acyl donors (I) and acceptors (II): as for I, propionyl-CoA was 1/10 as active as acetyl-CoA with L-leucine as the acceptor; as for II, L-leucyl-D-leucine, L-leucyl-L-leucine, L-arginine, L-leucyl-L-leucyl-L-leucine, L-phenylalanine, L-methionine, L-leucine, glycyl-L-phenylalanine and L-valine were acetylated in the decreasing order, as opposed to no or slight reactivity of D-phenylalanine, D-leucine, L-histidine, glycine, L-proline and L-glutamic acid. The Michaelis constants of acetyl-CoA and L-leucine were about 5 X 10(-6) M and 6 X 10(-5) M, respectively. The enzyme is stimulated by Fe++. p-Chloromercuribenzoic acid (PCMB), N-ethylmaleimide, Mg++ and Mn++ were inhibitory. A leupeptin-nonproducing mutant, Streptomyces roseus MA839-A1 LN-S, derived from the producer strain by acriflavine treatment, also produced this enzyme.

Biosynthesis of leupeptin. II Purification and properties of leupeptin acid synthetase.

An enzyme which condenses acetyl-L-leucyl-L-leucine and L-arginine into acetyl-L-leucyl-L-leucyl-L-leucyl-L-arginine (leupeptin acid) was partially purified from a cell extract of Streptomyces roseus MA839-A1. With respect to this catalytic activity, the enzyme showed the following characteristics: ATP is essential; optimum pH is 9.5; the activity is inhibited either by EDTA or pyrophosphate or N-ethylmaleimide. The molecular weight of the enzyme is about 260,000 daltons. It also catalyzes some other extension reactions, such as, acetyl-L-leucine+L-leucine+L-arginine leads to leupeptin acid, and acetyl-L-leucine+L-leucine leads to acetyl-L-leucyl-L-leucine, but neither L-leucine+L-arginine leads to (L-leucyl)1--2-L-argining, nor acetyl-L-leucine+L-arginine leads to acetyl-L-leucyl-L-arginine. ATP-PPi exchange, catalyzed by this enzyme, proceeds with either acetyl-L-leucine, or acetyl-L-leucyl-L-leucine or L-leucine, but not with acetate or arginine.

Biochemical study of minosaminomycin in relation to the kasugamycin group antibiotics.

Minosaminomycin is structurally related to kasugamycin and inhibits protein synthesis in mycobacteria. It also inhibits phage f2 RNA-directed protein synthesis in a cell-free system of Escherichia coli by 50% at 2 x 10(-7) M. It is 100-times more potent than kasugamycin in this system. At 10(-7) M minosaminomycin inhibits EF-T dependent binding of aminoacyl-tRNA to ribosomes by 50%. This effect is markedly diminished if minosaminomycin is added to the assay system after a brief incubation of ribosomes with mRNA. Like kasugamycin, minosaminomycin preferentially inhibits the initiation of protein synthesis directed by phage f2 RNA in vitro and does not cause miscoding. Ribosomes from kasugamycin-resistant mutants Ksg A and Ksg C were as sensitive to minosaminomycin as those from each parent strain. In spite of the strong inhibitory activity of minosaminomycin manifested in cell-free systems of E. coli, this compound inhibits the growth of the organism itself only slightly. This discrepancy could be ascribed to impermeability, as E. coli cells with modified permeability show greater sensitivity to minosaminomycin. There is no indication that the antibiotic is activated in E. coli cells. On the basis of these results, the structural features of these antibiotics is inactivated in E. coli cells. On the basis of these results, the structural features of these antibiotics essential for interaction with ribosomes and for permeability into the cells are discussed.