Synthesis of Valine and Isoleucine in the Presence of a Particulate Cell Fraction of Neurospora

Abstract

It has been established that valine is synthesized from pyruvate in a series of at least four steps, and that isoleucine is synthesized in a series of four similar steps from pyruvate and a-ketobutyrate. The four enzymes involved are apparently common to both pathways, but this has not been definitely proved in any one case. The enzymes will be described here as the condensing enzyme, which produces aacetolactate and a-acetohydroxybutyrate from pyruvate, and pyruvate + aketobutyrate, respectively; the reductoisomerase, which converts the a-aceto-acids to the dihydroxy acids; the dehydrase which causes a dehydration of the dihydroxy acids with the formation of the a-keto acids; and the transaminase which transaminates between phenylalanine and the respective a-keto acids to form valine and isoleucine.In at least two organisms, Salmonella and Neurospora, it has been shown that two or more of the genes affecting these enzymes are closely linked.1-3 In Neurospora crassa at least two of the enzymes, reductoisomerase and dehydrase, are apparently controlled by two genes which are estimated to be about four map units apart.4 Between them lies a region in which mutations have been detected only within a small segment. These mutations cause an absolute requirement for both isoleucine and valine, but the mutant strains do not show a deficiency for any of the four enzymes. That the blocks in these are not prior to pyruvate and aacetobutyrate has been established by showing that they accumulate either the a-aceto-acids or dihydroxy acids or both. It was postulated that the defect in these mutants, the group II mutants, lies not in their inability to produce the enzymes, but in their inability to organize them properly to be active in vivo.3 The experiments described below were designed to explore this possibility by first determining whether there exists an isoleucine-valine forming system in the particulate fraction of the wild-type mycelial homogenate.Methods and Materials.-The assay procedures for the individual enzymes, the condensing enzyme, reductoisomerase, dehydrase, and transaminase have been described in detail previously.'-Succinic cytochrome c reductase activity was determined by the method of Crane et al.9 Protein was determined by the method of Lowry et al.10The wild-type strain employed here, KJT196Oa, is a segregant from a cross between the Emer-

Antipolarity in the ilv Operon of Escherichia coli K-12

“…The antipolar effect of the E-amber mutation on DH is also consistent with this theory. It is noteworthy that certain enzymes of the isoleucine-valine biosynthetic pathway appear to be aggregated in N. crassa (34); nevertheless, we have been unable to demonstrate such aggregation in extracts of our wild-type E. coli strain. The three enzymes diffuse independently of each other, and TD and DH separate readily during ammonium sulfate fractionation.…”

SYNCHRONOUS MITOCHONDRIAL DIVISION IN NEUROSPORA CRASSA

“…Wild type mycelium of known age was harvested after 35 hr of growth from a minimal agar plate overlaid with dialysis membrane and was then fixed for electron microscopy. Seven samples were harvested at the same time from regions of a single mycelial pad known to be 4, 8,12,16,20,24, and 30 hr old.…”

Acetohydroxy Acid Synthetase with a p H Optimum of 7.5 from Neurospora crassa Mitochondria: Characterization and Partial Purification

“…The a-ketobutyrate inhibition of purified AAS was not unexpected. Wagner and co-workers (27) have shown that a-ketobutyrate will competitively inhibit valine synthesis from pyruvate as determined by in situ assay of the Cmp from Neurospora. The competitive inhibition of purified mitochondrial AAS is presumably due to the ability of a-ketobutyrate to occupy the active site(s) as a pyruvate analogue.…”