Studies on the Mode of Action of Polyoxins
1977 Volume 2 Issue 3 Pages 345-355
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1977 Volume 2 Issue 3 Pages 345-355
Polyoxins A-M, which belong to the class of peptidyl-pyrimidine nucleoside antibiotics, are produced by Streptomyces cacaoi var. asoensis. Polyoxins, except C and I, are active against various phytopathogenic fungi. Polyoxins A, B and D are shown to inhibit selectively the incorporation of 14C-glucosamine into cell wall chitin by washed mycelia of Alternaria kikuchiana, Cochliobolus miyabeanus and Pyricularia oryzae at levels which are comparable to their minimal inhibitory concentrations for the fungal frowth. At the same time, each active polyoxin causes an unusually increased accumulation of UDP-14C-N-acetylglucosamine (UDP-GlcNAc). Polyoxins A-M, except C and I, markedly inhibit chitin synthetase preparation from P. oryzae in competition with the substrate, UDP-GlcNAc. Michaelis constant, Km, for UDP-GlcNAc is 3.3×10-3M and inhibitor constant, Ki, for the active polyoxins is found to be in the range of 3.3×10-5-3.4×10-6M. At higher concentrations such as 3mM, polyoxin C also competitively inhibits the enzyme, and the Ki is close to the Km. It is concluded that polyoxins selectively inhibit the synthesis of cell wall chitin and that the primary effect is the competitive inhibition of chitin synthetase. To correlate the polyoxin structure to the enzyme inhibition, the values of binding affinity, -ΔGbind*, for polyoxins and various N-aminoacyl derivatives of polyoxin C are calculated from the Ki values, and the values of partial binding affinity, -Δg*, for structural moieties which constitute the polyoxin J molecule are estimated from the -ΔGbind values. The results indicate that the carbamoylpolyoxamic acid moiety of polyoxins serves to stabilize the complexing of polyoxin with the enzyme. The same kinetic study using nucleosides, nucleotides and nucleotide sugars suggests the presence of a specific binding site on the enzyme for the uridine moiety of UDP-GlcNAc, where the pyrimidine nucleoside moiety of polyoxins also binds. From the pKi-pH plots for the derivatives of polyoxin C and other polyoxins, it is found that the ionized amino group at C-2″ position plays a very important role for the binding of polyoxins to chitin synthetase.
* -ΔG=-RTlnKi
-ΔGbind=-ΔG+2.4
-ΔGbind=-(Δg′+Δg″+Δg′′′+…+Δgn) (kcal/mol, 25°C)
