Makoto N ISHIYAMA Biotechnology Research Center, The University of Tokyo
Research aims
We found that the extremely thermophilic bacterium, Thermus thermophilus HB27 synthesizes lysine via non-di- aminopimelate pathway ( α -aminoadipate pathway). α- Aminoadipate pathway is known to be present only in lower eukaryotes, such as fungi and yeast, and Thermus lysine pathway proceeds in the same way until α -aminoadipate synthesis. However, the synthesis from α-aminoadipate to lysine in Thermus pathway is different from the pathway in lower eukaryote with saccharopin as a biosynthetic inter- mediate, and therefore the pathway is novel as a whole. The reactions from 2-oxoglutarate to α-aminoadipate in the pathway is similar to those from 2-oxoisovalerate to leucine in leucine biosynthesis and from oxaloacetate to 2-oxoglu- tarate in the tricarboxylic acid cycle. Furthermore, the reac- tions from α -aminoadipate to lysine proceeds in a way sim- ilar to those from glutamate to ornithine in arginine biosyn- thesis, and some reactions in biosynthesis of proline and as- partate group amino acids. Thus, the lysine biosynthesis is evolutionarily related to other biosynthetic or metabolic pathways. These observations suggest that the lysine biosynthesis or its ancestral pathway had a definitive role in developing biological system in the early stage of evolution. This study aims at i) elucidation of the substrate specificity, reaction mechanism, and feedback regulation system of the newly found lysine biosynthesis of T. thermophilus , and ii) establishment of the base for new lysine fermentation process.
Methods
We have developed the system to express the genes in- volved in the lysine biosynthesis from T. thermophilus HB27 in Escherichia coli cells. After purification of the en- zymes, their catalytic properties were analyzed by using several related compounds; those are intermediates in argi- nine and leucine biosynthesis and in the tricarboxylic acid cycle. Based on the information on the primary and tertiary structures of related enzymes, amino acid residues that de- termine substrate specificity were chosen as candidates, and their contribution was analyzed by site-directed mutagene- SIS.
Results
Our previous studies revealed that lysine is synthe- sized from 2-oxoglutarate through ten steps of the reac- tions. In the present study, we have tried to clone two genes, homoisocitrate dehydrogenase converting homoisocitrate to α-ketoadipate and α-aminoadipate aminotransferase cat- alyzing the following reaction to produce α -aminoadipate, which have not yet been cloned. Since the former gene was expected to have the amino acid sequence homologous to those of isocitrate dehydrogenase and isopropylmalate de- hydrogenase, we used degenerate primers designed to cor- respond to internal sequences for PCR cloning. The latter gene was also cloned by PCR based on the primary se- quence of the mammalian enzyme that was reported to have ability to use α -aminoadipate as a amino donor. We gener- ated Thermus mutants carrying disruption of both the genes and examined their auxotrophy. Disruptant of homoisoci- trate dehydrogenase gene exhibited complete lysine-aux- otrophic phenotype and disruptant of α-aminoadipate aminotransferase gene showed partial lysine-auxotrophic phenotype, on minimal medium. Homoisocitrate dehydro- genase produced in E. coli cells were characterized by using homoisocitrate or its structurally related compounds. We have succeeded in identification of amino acid residues that determine the substrate specificity by site-directed mu- tagenesis based on the three dimensional structures of isoc- itrate dehydrogenase and isopropylmalate dehydrogenase, both of which share reaction mechanism and substrate recognition mechanism with homoisocitrate dehydrogenase. As to α-aminoadipate aminotransferase, we are obtaining preliminary results that the enzyme has branched chain amino acid aminotransferase activity, though no significant identity in amino acid sequence is observed between Ther- mus α-aminoadipate aminotransferase and branched chain amino acid aminotransferase. It is well known that an enzyme responsible for the first reaction in amino acid biosynthetic pathway is regu- lated via feedback inhibition by end product of the pathway. We therefore analyzed homocitrate synthase that catalyzes the first reaction in the lysine biosynthesis through α- aminoadipate, and found that the enzyme was inhibited by lysine with high sensitivity ( K i = 9.4 µ M). Homocitrate syn- thase could not recognize 2-oxoisovalerate as a substrate though the synthase has the amino acid sequene similar to that of isopropylmalate dehydrogenase. However, the en- zyme could utilize oxaloacetate as a substrate, although no sequence homology was found between homocitrate syn- thase and citrate synthase. This suggests that homocitrate synthase also serves as citrate synthase in Thermus cells under some circumstance.
Conclusion
We have succeeded in cloning all the genes involved in lysine biosynthesis from T. thermophilus HB27, and have developed their efficient expression system in E. coli. Sev- eral information on the enzymatic properties including sub- strate recognition is being accumulated. We therefore think that we have successfully developed the base for lysine pro- duction through the studies. We will move on to establish- ment of lysine fermentation process based on the accumu- lated information.
Reference
Miyazaki, J., Kobashi, N., Nishiyama, M., and Yamane, H. Characteriza- tion of homoisocitrate dehydrogenase involved in lysine biosynthesis of an extremely thermophilic bacterium, Thermus thermophilus HB27, and evolutionary implication of β -decarboxylating dehydrogenase. J. Biol. Chem., 278, 1864–1871 (2003)