In Saccharomyces cerevisiae, selection for resistance to the leucine analog 5',5',5'-trifluoroleucine (TFL), resulted in the identification of two genetically distinct groups of mutants, one comprising all the dominant (TFL1) and the other one all the recessive (tfl2) mutations (Casalone et al, 1997). The dominant mutations are located on the LEU4 gene, encoding alpha-isopropylmalate synthase I (2-isopropylmalate synthase) (Casalone et al, 1997). These mutations resulted in accumulation of leucine as a consequence of the synthesis of an enzyme insensitive to feedback inhibition by leucine (Casalone et al, 1997). In certain of the mutants the enzyme was resistant to Zn²⁺-mediated inactivation by Coenzyme A (Cavalieri et al, 1999).

Yeast (Saccharomyces cerevisiae) strains that bear disruption alleles of LEU4, are Leu+ and exhibit a level of alpha-isopropylmalate synthase activity that is 20% of the wild type (Drain and Schimmel, 1988). The leu4 disruption strain permitted identification of three new complementation groups, designated leu6, leu7 and leu8 (Drain and Schimmel, 1988). Each of these new mutations effect leucine auxotrophy only if a leu4 mutation is present and each results in loss of alpha-IPM synthase activity (Drain and Schimmel, 1988). LEU7 and LEU8 are candidates for the gene or genes that encode a second isoform of alpha-IPM synthase (Drain and Schimmel, 1988).

Little is known about the corresponding gene(s) encoding 2-isopropylmalate synthase from higher plants. However, this first enzyme of the leucine biosynthesis pathway is strongly feedback-regulated by leucine in spinach chloroplasts (Hagelstein and Schultz, 1993).

In the yeast Saccharomyces cerevisiae, isopropylmalate isomerase is encoded by the LEU1 gene (Skala, 1991), whereas beta-isopropylmalate dehydrogenase (3-isopropylmalate dehydrogenase) is encoded by LEU2 (Brisco et al, 1987). Aspergillus niger possesses two highly divergent, differentially regulated, isozymes for beta-isopropylmalate dehydrogenase encoded by leu2A and leu2B (Williams et al, 1996).

Beta-isopropylmalate dehydrogenase (3-isopropylmalate dehydrogenase) (IPMDH) has been intensively investigated in microorganisms. IPMDH and isocitrate dehydrogenase (ICDH) belong to a unique family of bifunctional decarboxylating dehydrogenases. The crystal structure of IPMDH has been determined for Escherichia coli, Salmonella typhimurium, Thermus hermophilus, and Thiobacillus ferrooxidans (Wallon et al, 1997; Imada et al, 1998). The thermal stability of the enzyme from Bacillus subtilis was improved by an in vivo evolutionary technique using the extreme thermophile, Thermus thermophilus, as a host cell (Akanuma et al, 1998; 1999). The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus, and mutants were then selected which showed a leucine-autotrophy at progressively higher selection temperatures (Akanuma et al, 1998). DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine (Akanuma et al, 1998). The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme, and the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme (Akanuma et al, 1998). Additional thermostabilizing substitutions were subsequently found by site-directed mutagenesis (Akanuma et al, 1999).

3-Isopropylmalate dehydrogenase [EC 1.1.1.85] appears to be encoded by a single gene in both oilseed rape (Ellerstrom et al, 1992) and potato (Jackson et al, 1993). The rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) was cloned by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The protein is imported into chloroplasts in vitro concomitantly with a proteolytic cleavage (Ellerstrom et al, 1992). The potato cDNA also reveals a putative chloroplast transit peptide, suggesting chloroplast/plastid localization of the pathway (Jackson et al, 1993; Singh and Shaner, 1995).

As for valine and isolecine synthesis (see preceding page), the terminal step in the leucine biosynthesis pathway involves transamination catalyzed by leucine aminotransferase [EC 2.6.1.6].

The Leu3 protein of Saccharomyces cerevisiae is a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways (Hu et al, 1995). Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. Activation by Leu3 requires the presence of alpha-isopropylmalate (Hu et al, 1995). Leu3 also interacts with the UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase (Hu et al, 1995). Thus, GDH1, which plays an important role ammonia assimilation in yeast cells, is, in part, activated by the Leu3-alpha-isopropylmalate complex (Hu et al, 1995).

In Neurospora crassa alpha-isopropylmalate and the leu-3 product may interact to regulate enzymes of the histidine pathway (Kidd and Gross, 1984).

Akanuma S, Yamagishi A, Tanaka N, Oshima T 1998 Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution. Protein Sci. 7: 698-705.

Akanuma S, Yamagishi A, Tanaka N, Oshima T 1999 Further improvement of the thermal stability of a partially stabilized Bacillus subtilis 3-isopropylmalate dehydrogenase variant by random and site-directed mutagenesis. Eur. J. Biochem. 260: 499-504.

Brisco PR, Cunningham TS, Kohlhaw GB 1987 Cloning, disruption and chromosomal mapping of yeast LEU3, a putative regulatory gene. Genetics 115: 91-99.

Casalone E, Fia G, Barberio C, Cavalieri D, Turbanti L, Polsinelli M 1997 Genetic and biochemical characterization of Saccharomyces cerevisiae mutants resistant to trifluoroleucine. Res. Microbiol. 148: 613-623.

Cavalieri D, Casalone E, Bendoni B, Fia G, Polsinelli M, Barberio C 1999 Trifluoroleucine resistance and regulation of alpha-isopropyl malate synthase in Saccharomyces cerevisiae. Mol. Gen. Genet. 261: 152-160.

Drain P, Schimmel P 1988 Multiple new genes that determine activity for the first step of leucine biosynthesis in Saccharomyces cerevisiae. Genetics 119: 13-20.

Ellerstrom M, Josefsson LG, Rask L, Ronne H 1992 Cloning of a cDNA for rape chloroplast 3-isopropylmalate dehydrogenase by genetic complementation in yeast. Plant Mol. Biol. 18: 557-566.

Hagelstein P, Schultz G 1993 Leucine synthesis in spinach chloroplasts: partial characterization of 2-isopropylmalate synthase. Biol. Chem. Hoppe. Seyler 374: 1105-1108.

Hu Y, Cooper TG, Kohlhaw GB 1995 The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation. Mol. Cell Biol. 15: 52-57.

Imada K, Inagaki K, Matsunami H, Kawaguchi H, Tanaka H, Tanaka N, Namba K 1998 Structure of 3-isopropylmalate dehydrogenase in complex with 3-isopropylmalate at 2.0 A resolution: the role of Glu88 in the unique substrate-recognition mechanism. Structure 6: 971-982.

Jackson SD, Sonnewald U, Willmitzer L 1993 Cloning and expression analysis of beta-isopropylmalate dehydrogenase from potato. Mol. Gen. Genet. 236: 309-314.

Kidd GL, Gross SR 1984 Specific regulatory interconnection between the leucine and histidine pathways of Neurospora crassa. J. Bacteriol. 158: 121-127.

Bryan JK 1980 Aspartate family and branched-chain amino acids. In (BJ Miflin ed) "The Biochemistry of Plants", Vol. 5, Academic Press, New York, pp. 403-452.

Singh BK, Shaner DL 1995 Biosynthesis of branched chain amino acids: From test tube to field. Plant Cell 7: 935-944.

Skala J, Capieaux E, Balzi E, Chen WN, Goffeau A 1991 Complete sequence of the Saccharomyces cerevisiae LEU1 gene encoding isopropylmalate isomerase. Yeast 7: 281-285.

Wallon G, Kryger G, Lovett ST, Oshima T, Ringe D, Petsko GA 1997 Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus. J. Mol. Biol. 266: 1016-1031.

Williams BA, Sillaots S, Tsang A, Storms R 1996 Isolation by genetic complementation of two differentially expressed genes for beta-isopropylmalate dehydrogenase from Aspergillus niger. Curr. Genet. 30: 305-311.