DNA-directed in vitro synthesis of Escherichia coli beta-isopropylmalate dehydrogenase.

The in vitro synthesis of beta-isopropylmalate dehydrogenase (EC 1.1.1.85), an enzyme involved in leucine biosynthesis, has been obtained using as template DNA from the hybrid plasmid (pLC1) which contains the Escherichia coli leucine operon. Enzyme synthesis in vitro is stimulated about 2-fold by guanosine-5'-diphosphate-3'-diphosphate and inhibited about 60% by 2 X 10(-4) M L-leucine.


SUMMARY
The in vitro synthesis of /Sisopropylmalate dehydrogenase (EC 1.1.1.85), an enzyme involved in leucine biosynthesis, has been obtained using as template DNA from the hybrid plasmid (pLC1) which contains the Escherichia coli leucine operon. Enzyme synthesis in vitro is stimulated about 2-fold by guanosine-5'-diphosphate-3'-diphosphate and inhibited about 60% by 2 x 10e4 M L-leucine.
It is now well established that the regulation of the synthesis of the enzymes for biosynthesis of the branched chain amino acids is regulated by multivalent repression (1,2). As shown in Fig. 1, the pathways for the synthesis of isoleucine, valine, and leucine have many steps in common and in the presence of all three amino acids, the gene products of the isoleucine-valine gene cluster and leucine operon are repressed.
In addition, there is considerable evidence that the cognate tRNAs are involved in the repression of these enzymes (3,4 coli JA219 strain, 2 mM NAD, and 6 pg of pLC1 DNA or as indicated. One hour of incubation at 37°C was used for enzyme synthesis after which chloramphenicol (2 pg) was added to the reaction mixtures to stop protein synthesis and 20 nmol of p-[U-"CIIPM (5000 cpm/nmol) were also added. P-IPM dehydrogenase activity was then determined in a second incubation by measuring the release of CO, from p-t U-14C]IPM. The assay tubes were incubated at 37°C for an additional 2 h, and at the end of the incubation, the reaction was terminated by the addition of 10 ~1 of 1 N HCl. After further incubation for 30 min, "CO, trapped in the ethanolamine/2-methoxyethanol mixture was placed in scintillation fluid (10) and the radioactivity was determined in a Beckman liquid scintillation spectrometer. All samples were corrected for a blank of 50 cpm that was obtained without added DNA.
Identification of cr-Ketoisocaproic acid -At the end of the incubation, the reaction was terminated by the addition of 10 ~1 of 1 N HCl and 0.15 ml of water. a-Ketoisocaproate (10 Kg) and 5 ~1 of 0.5% 2,4dinitrophenylhydrazine in 6 N HCl were added to the deproteinized sample. The mixture was allowed to react at room temperature for 16 h and then extracted with three 0.25-ml portions of chloroform, containing 20% ethanol. The solvent was evaporated to a small volume and subjected to cellulose thin layer chromatography in a solvent system containing methanol:benzene:butanol-1:water (4:2:2:2). In this solvent system, the R,,. values of P-IPM and the 2,4-dinitrophenylhydrazine of cu-ketoisocaproic acid are 0.80 and 0.89, respectively RESULTS DNA-directed S.ynthesis of /3-IPM Dehydrogenase -As described under "Materials and Methods," a two-step procedure was used; the first incubation to synthesize P-IPM dehydrogenase and the second to assay the amount of enzyme formed. More detailed kinetics of these two reactions are shown in Fig.   2. It is seen that enzyme synthesis is linear for 1 h but abruptly stops thereafter ( Fig. 2A)  but not NADP. The NAD-dependent release of CO, from p-IPM indicates that /3-IPM dehydrogenase is being synthesized (111. In addition, the radioactive product of the P-IPM dehydrogenase reaction was identified as a-ketoisocaproic acid by thin layer chromatography of its 2,4-dinitrophenylhydrazine derivative (see "Materials and Methods"). Effect of CAMP and ppGpp on the in Vitro Synthesis of /3-ZPM Dehydrogenase-As shown in Table II, CAMP had no effect on the synthesis of P-IMP dehydrogenase, whereas ppGpp gave a 2-to 3-fold stimulation of enzyme synthesis when added during the first incubation. There was no effect of ppGpp on P-IPM dehydrogenase activity, indicating that ppGpp stimulates the synthesis of /3-IPM dehydrogenase.
Effect of L-Leucine on in Vitro Synthesis-It has been shown in vivo that the leucine biosynthetic enzymes are repressed by leucine. An effect of leucine on P-IPM dehydrogenase can also be seen in vitro although the situation is complicated by the fact that L-leucine is also required for In Vitro Synthesis of Plsopropylmalate Dehydrogenase The assays were performed as described in the text except for the addition of various amounts of L-leucine, L-arginine, or o-leucine. In Experiment 1, L-leucine (0.055 or 0.220 rn& was added to the incubations as indicated. In Experiments 2 and 3, the reaction mixtures contained 0.055 rnM L-leucine and the concentrations of Larginine and o-leucine as indicated. synthesis of the enzyme. Fig. 3 shows the effect of leucine concentration on the synthesis of P-IPM dehydrogenase. In the absence of added leucine, there is some synthesis of ,9-IPM dehydrogenase due to the presence of endogenous leucine in the S-30 extract. The addition of exogenous L-leucine, up to a concentration of 5.5 x 1O-5 M, stimulates /3-IPM dehydrogenase synthesis. However, the synthesis of p-IPM dehydrogenase declined at L-leucine concentrations higher than 5.5 x 10m5 M and at 2 x 1O-4 M L-leucine, enzyme synthesis was inhibited about 60%. These concentrations of leucine did not affect p-IPM dehydrogenase activity or the in vitro synthesis of pgalactosidase (data not shown). Similar concentrations of Larginine or n-leucine did not inhibit P-IPM dehydrogenase synthesis (Table III). DISCUSSION The present report shows that the synthesis of P-IPM dehydrogenase can be obtained in an in vitro system directed by a hybrid plasmid DNA (pLC1) containing the Zeu operon. Similar to other operons involved in amino acid biosynthesis such as the try operon (12, 131, his operon (141, and arg A system (15), the in vitro synthesis of ,!?-IPM dehydrogenase was not affected by CAMP but was stimulated by ppGpp. The stimulation of a leucine biosynthetic enzyme by ppGpp is in basic agreement with the hypothesis of Stephens et al. (14) who proposed a unifying role of ppGpp as a signal molecule (alarmone) which senses an amino acid deficiency and redirects the cell's economy in response. One exception is the arg E system (131, in which the expression of the arg E gene was inhibited by ppGpp. The concentration of leucine used in the in vitro studies is crucial since leucine is exerting a dual role. It is required for the synthetic process but at higher concentrations, represses the synthesis of @-IPM dehydrogenase. The repression of p-IPM dehydrogenase synthesis by leucine in vitro is consistent with the in uiuo studies of Umbarger (1,2). The levels of the enzymes required for the synthesis of the branched chain amino acids are probably regu!ated by the protein product of the uzl gene (1,16) and leucine is known to be involved in this repression. In addition, leucyl-tRNA or leucyl-tRNA synthetase (or both) have been postulated to be required for the in uiuo repression of the leu operon (l-4). With the use of S-30 extracts from mutants which are no longer repressed by leucine (17,181, or by employing a more defined in vitro system (191, it may be possible to isolate the gene product(s) involved in the regulation of leucine biosynthesis and study the role of aminoacyl-tRNA in this process. This cannot be done with the present DNA-directed system because these components are present in the crude extract.