Proof of De Novo Synthesis of the Qa Enzymes of Neurospora Crassa During Induction

In Neurospora crassa three inducible enzymes are necessary to catabolize quinic acid to protocatechuic acid. The three genes encoding these enzymes are tightly linked on chromosome VII near methionine-7 (me-7). This qa cluster includes a fourth gene, qa-1, which encodes a regulatory protein apparently exerting positive control over transcription of the other three qa genes. However, an alternative hypothesis is that the qa-1 protein simply activates preformed polypeptides derived from the three structural genes. The use of density labeling with D(2)O demonstrated conclusively that the qa enzymes are synthesized de 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 ca 22 identical subunits) has a density of 1.2790 g/cm(3) as determined by centrifugation in a modified cesium chloride density gradient. Growth in H(2)O followed by induction in 95% D(2)O shifts the density of the enzyme to 1.3130 g/cm(3), indicating de novo synthesis during induction. In the reciprocal experiment, i.e., growth in 80% D(2)O followed by induction in either 95% D(2)O or H(2)O, the densities of catabolic dehydroquinase were 1.3135 and 1.2800 g/cm(3), respectively. Because growth on D(2)O does not affect the density of the H(2)O-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.