Proof of de novo synthesis of the qa enzymes of Neurospora crassa during induction

Abstract

In N. crassa 3 inducible enzymes are necessary to catabolize quinic acid to protocatechuic acid. The 3 genes encoding these enzymes are tightly linked on chromosome VII near methionine-7(me-7). This qa cluster includes a 4th gene, qa-1, which encodes a regulatory protein apparently exerting positive control over transcription of the other 3 qa genes. An alternative hypothesis is that the qa-1 protein simply activates preformed polypeptides derived from the 3 structural genes. The use of density labeling with D2O demonstrated conclusively that the qa enzymes are synthesized do novo only during induction on quinic acid. Native catabolic dehydroquinase (5-dehydroquinate dehydratase; 5-dehydroquinate hydro-lyase, EC 4.2.1.10)(a homopolymer of about 22 identical subunits) has a density of 1.2790 g/cm3 as determined by centrifugation in a modified cesium chloride density gradient. Growth in H2O followed by induction in 95% D2O shifts the density of the enzyme to 1.3130 g/cm3, indicating de novo synthesis during induction. In the reciprocal experiment, i.e., growth in 80% D2O followed by induction in either 95% D2O or H2O, the densities of catabolic dehydroquinase were 1.3135 and 1.2800 g/cm3, respectively. Because growth on D2O does not affect the density of the H2O-induced enzyme, there can be no significant synthesis of catabolic dehydroquinase prior to induction. Similar results were obtained for a second qa enzyme, quinate dehydrogenase (quinate:NAD+ oxidoreductase, EC 1.1.1.24). Thus, induction of two qa enzymes involves de novo protein synthesis, not enzyme activation or assembly.