Isoleucine Hydroxamate, an Isoleucine Antagonist

Abstract

Isoleucine hydroxamate (Ile Hdx) was found to inhibit the growth of Serratia marcescens and to antagonize isoleucine. At a low concentration of Ile -Hdx, at which the growth of the wild strain was completely inhibited, the growth of an isoleucine auxotroph was not inhibited in the medium containing a limiting amount of D-threonine as the isoleucine source. At a higher concentration, this antagonist exhibited a considerable inhibitory effect on the growth of the auxotroph. IleoHdx showed the same inhibitory effect as isoleucine on L-threonine dehydratase activity at the concentrations 10 times those of isoleucine. Ile Hdx caused also derepression of isoleucine-valine biosynthetic enzymes and the derepression was overcome by isoleucine. These results indicate the Ile* Hdx causes growth inhibition by its effects on isoleucine metabolism.In the previous papers (13,14), it was reported that a-aminobutyric acid-resistant mutants of Serratia marcescens accumulated valine and accumulated isoleucine derived from D-threonine. Further investigation revealed some features of the regulatory mechanisms for isoleucine-valine biosynthesis in the wild strain and the basis of the genetic derepression of isoleucinevaline biosynthetic enzymes in the mutants (14a).It has been generally accepted that wild-type organisms do not accumulate isoleucine because of feedback control of L-threonine dehydratase. In view of the results on isoleucine accumulation in the presence of D-threonine (12, 13), it was anticipated that isoleucine would be overproduced from L-threonine by regulatory mutants of S. marcescens that lack feedback inhibition of Lthreonine dehydratase by isoleucine. We considered that mutants lacking such feedback inhibition might be found among the class of mutants resistant to an analogue of isoleucine. Although cyclopentaneglycine, cyclohexene-1-glycine, and thiaisoleucine are known as isoleucine antagonists in some bacteria (3,4,9,16,19), a resistant mutant with an isoleucine-insensitive Lthreonine dehydratase has not been obtained.For these reasons, we attempted to search for other isoleucine antagonists in S. marcescens. Of several isoleucine analogues tested, isoleucine hydroxamate (Ile Hdx) was found to inhibit CH3-CH2 NH ,. CH-CH-COOH CH3 Isolcucine CH3-CH2 NH2 , CH-CH-CONHOH CH3 Isoleucine hydroxomate (11e-Hdx)growth of S. marcescens. Ile*Hdx differs from isoleucine in having a CONHOH group instead of a COOH group. This paper deals with the bacteriostatic action of Ile Hdx. A separate paper will describe the properties of mutants resistant to this antagonist and the accumulation of isoleucine by feedback inhibition-negative mutants in L-threonine-containing media. MATERIALS AND METHODSBacteria. The bacterial strains used in this study were S. marcescens strain I (11) and the isoleucine auxotroph strain 420 derived from it.Growth experiments. Cultures were grown in test tubes (13 by 103 mm) containing 2 ml of Davis-Mingioli minimal medium (2) modified by omitting the citrate and increasing the glucose to 0.5%. Suppleme...

In vitro formation of beta-galactosidase with a template containing the lac genes fused to gene ilvD

“…Several -8 amino acid hydroxamates have been shown to z inhibit amino acyl tRNA synthetases (28). Al-JI though isoleucine hydroxamate has not been 25 thoroughly investigated in E. coli, a Serratia marcescens culture grown in its presence exhibits a derepression of the ilvEDA operon (12). dlacZp was the template employed, but signifimixtures contained isoleucine hydroxamate as indicantly less inhibition was seen when pPU34 was cated.…”

“…A reduced rate of protein synthesis was achieved by using two antimetabolites that are thought to interfere with the formation of isoleucyl tRNA, pseudomonic acid and isoleucine hydroxamate (8,12). With each agent, overall protein synthesis was retarded, as was synthesis of f8-galactosidase.…”

Threonine degradation by Serratia marcescens

“…Cells were harvested from exponentially growing culture by centrifugation and washed twice with 50 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 8.5). Cells were suspended in the same buffer, and cell-free extracts were prepared as described previously (14). The extracts prepared thus were used for the assay of threonine dehydrogenase and threonine aldolase.…”

“…The extracts prepared thus were used for the assay of threonine dehydrogenase and threonine aldolase. Cell-free extracts for threonine deaminase were prepared by the method stated previously (14).…”

“…Threonine deaminase was assayed as described previously (14), except that the reaction mixture was incubated at 300C. Specific activities are expressed as micromoles of products per milligram of protein per minute.…”