Effect of mutations in the qa gene cluster of Neurospora crassa on the enzyme catabolic dehydroquinase

Abstract

Catabolic dehydroquinase, which functions in the inducible quinic acid catabolic pathway of Neurospora crassa, has been purified from wild type (74-A) and three mutants in the qa gene cluster. The mutant strains were: 105c, a temperature-sensitive constitutive mutant in the qa-1 regulatory locus; M-16, a qa-3 mutant deficient in quinate dehydrogenase activity; and 237, a leaky qa-2 mutant which possess very low levels of catabolic dehydroquinase activity. The enzymes purified from strains 74-A, 105c, and M-16 are identical with respect to behavior during purification, specific activity, electrophoretic behavior, stability, molecular weight, subunit structure, immunological cross-reactivity, and amino acid content. The mutant enzyme from strain 237 is 1,500-fold less active and appears to have a slightly different amino acid content. It is identical by a number of the other criteria listed above and is presumed to be a mutant at or near the enzyme active site. These data demonstrate that the qa-1 gene product is not involved in the posttranslational expression of enzyme activity. The biochemical identity of catabolic dehydroquinase isolated from strains 105c and M-16 with that from wild type also demonstrates that neither the inducer, quinic acid, nor other enzymes encoded in the qa gene cluster are necessary for the expression of activity. Therefore the combined genetic and biochemical data on the qa system continue to support the hypothesis that the qa-1 regulatory protein acts as a positive initiator of qa enzyme synthesis.

Gene organization and regulation in the qa (quinic acid) gene cluster of Neurospora crassa.

“…(i) C-DHQase. C-DHQase (encoded by the qa-2+ gene) was further characterized in studies of the purified enzyme from the wild type, certain qa-2 mutants, and other mutants with mutations in other genes (qa-Jc and qa-3-) of the qa cluster (10,26,30). These studies concluded that the purified wild-type enzyme possesses several unusual structural, physical, and biochemical properties, e.g., a very high specific activity, a high degree of stability in the presence of a variety of denaturing conditions (including high temperatures) and reagents, and a polymeric structure with a molecular weight of ca.…”

Characterization of qa-2 mutants of Neurospora crassa by genetic, enzymatic, and immunological techniques

“…The fourth gene, qa-1, encodes a regulatory protein that, in the presence of inducer (quinic acid), acts in a positive fashion to initiate coordinate synthesis of the three qa enzymes (3)(4)(5)(6). Evidence supporting this hypothesis has come from comparative studies of purified catabolic dehydroquinase (7) obtained from the wild type and from mutants bearing lesions in the qa gene cluster (9). These studies indicate that the properties of purified catabolic dehydroquinase obtained from the wild type and from mutants with lesions in the qa-1 gene or the qa-3 gene are identical, as predicated by the hypothesis.…”

“…These studies indicate that the properties of purified catabolic dehydroquinase obtained from the wild type and from mutants with lesions in the qa-1 gene or the qa-3 gene are identical, as predicated by the hypothesis. In contrast, the catabolic dehydroquinase from one qa-2 mutant (M237) was found to be 1,500-fold less active and appeared to have a slightly different amino acid content (9). Despite the low activity, this mutant was shown to be immunologically competent in tests with antisera against catabolic dehydroquinase and to have wild-type levels of catabolic dehydroquinase protein.…”