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.     

Genetic Fine Structure of the Leucine Operon of Escherichia coli K-12

The order of mutational sites in 10 independently isolated leucine auxotrophys of Escherichia coli K-12 was determined by three-point reciprocal transductions. The sites of mutation mapped in linear sequence in a cluster; all leucine auxotrophic mutations were cotransducible with mutations in the arabinose operon. The mutations were assigned to four complementation groups by abortive transduction tests, designated D, C, B, and A, reading in a clockwise direction from the arabinose operon. Enzyme analyses showed that strains with a mutation in gene A lacked alpha-isopropylmalate synthetase activity (EC 4.1.3), and those with a mutation in gene B lacked beta-isopropylmalate dehydrogenase activity (EC 1.1.1). It is concluded that the gross structure of the leucine operon in E. coli is closely similar to, if not identical with, the gross structure of the leucine operon in Salmonella typhimurium.     

Importance of the Isovalerate Carboxylation Pathway of Leucine Biosynthesis in the Rumen

Certain anaerobic ruminal bacteria synthesize the leucine carbon skeleton by use of a pathway different from that described in other microorganisms. These organisms carboxylate the intact carbon skeleton of isovalerate, synthesizing leucine-2-C(14) from isovalerate-1-C(14). Strains of Bacteroides ruminicola and Peptostreptococcus elsdenii were like Ruminococcus flavefaciens in that they incorporated appreciable amounts of C(14) from isovalerate-1-C(14) into cellular protein and in that the only labeled amino acid found was leucine. The specific activity of beta-isopropylmalate dehydrogenase in extracts from R. flavefaciens and from the mixed bacterial population from the rumen was very low as compared with the specific activity of this enzyme in extracts from Escherichia coli. This suggests that the pathway of leucine biosynthesis that operates in many aerobic and facultative microorganisms is not the major pathway in rumen bacteria. This was supported by the finding that after fermentation of whole rumen contents with acetate-2-C(14), leucine from the bacterial cells had a specific activity lower than one would expect if acetate was incorporated directly into carbons 1 and 2 of leucine.     

Biosynthesis of Branched-Chain Amino Acids in Yeast: Correlation of Biochemical Blocks and Genetic Lesions in Leucine Auxotrophs

The three enzymatic steps in the conversion of alpha-ketoisovalerate to alpha-ketoisocaproate were examined in wild-type and in leucine auxotrophic stocks of yeast. Procedures for the reliable assay of each of the enzymatic steps in crude extracts were devised. Crude extracts of the prototrophic haploid stock catalyzed all three enzymatic steps. Examination of a series of leucine auxotrophs permitted a correlation between the three enzymatic steps and the genetic lesions affecting 10 different loci. This examination revealed that a single locus (le-6) affected primarily alpha-isopropylmalate synthetase, the first step in the pathway. Lesions in six loci (le-1, le-4, le-5, le-7, le-8, and le-10) lead primarily to a deficiency in the activity of the second enzyme in the pathway, alpha-isopropylmalate isomerase. Stocks with lesions in three loci (le-2, le-3, and le-9) were primarily blocked in the third step of the pathway, catalyzed by beta-isopropylmalate dehydrogenase. The results with the mutants provide strong evidence that the pathway for leucine biosynthesis proposed by Strassman and his colleagues is the sole significant pathway in yeast.     

Isoleucine biosynthesis in Leptospira interrogans serotype lai strain 56601 proceeds via a threonine-independent pathway

Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded alpha-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower K(m) (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed beta-methyl-d-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.     

Production of l-Lysine by Leucine Auxotrophs Derived from AEC Resistant Mutant of Brevibacterium lactofermentum

The effect of amino acids was examined on the production of l-lysine by AEC resistant mutant of B. lactofermentum. Among amino acids tested, only leucine showed strong specific inhibition. In order to release the production of l-lysine from this negative effect of leucine, leucine auxotrophs were derived from AEC resistant strain of B. lactofermentum. Most of these leucine auxotrophs produced larger amount of l-lysine (maximally 41 mg/ml) than the parental strain which produced about 18 mg/ml of l-lysine. It was confirmed that leucine auxotrophs derived from AEC resistant mutant of other glutamate producing bacteria, B. saccharolyticum and Corynebacterium glutamicum. These results suggested that leucine might directly or indirectly affect the biosynthesis of lysine.

However, this increase in lysine productivity of leucine auxotrophs could not be explained by the alteration of aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3). These enzymes are key enzymes in lysine and threonine biosynthesis, respectively.     

Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid

Escherichia coli harboring a recombinant plasmid was cultivated in fed-batch culture to enhance production of a gene product. Expression of the leucine gene from Thermus thermophilus in the recombinant plasmid was examined by the assay of beta-isopropylmalate dehydrogenase activity at 75 degrees C. When E. coli was cultivated in medium without leucine, biomass concentration reached 15 g/L and the specific activity became 0.082 U/mg protein. When leucine was fed in the medium throughout cultivation, although biomass concentration reached 63 g/L, the specific activity decreased to 0.016 U/mg protein. When E. coli was cultivated in medium containing 1 g leucine/L, the specific activity remained virtually constant (about 0.13 U/mg protein) and biomass concentration reached 32 g dry cells/L. In these cultivations, growth yields of several amino acids and glucose were examined. When leucine was not added to the medium, growth yields except for histidine were lowest. When leucine was fed throughout the cultivation, growth yields of glucose and tryptophan were highest. The pH-stat was useful for feeding amino acids.     

Genetics of leucine biosynthesis in Bacillus megaterium QM B1551.

Genes involved in the biosynthesis of leucine have been mapped in Bacillus megaterium QM B1551, using transducing phage MP13. Mutations were designated leuA, leuB, or leuC on the basis of enzyme assays. Two mutant strains were deficient in the enzyme activities of leuA (alpha-isopropylmalate synthase) and leuC (beta-isopropylmalate dehydrogenase) and so may contain polar mutations. Fine-structure transduction mapping established the gene order leuC-leuB-leuA-ilv-hem-phe. The orientation of the leu genes to the ilv gene is the same as in Bacillus subtilis, but the relationship in respect to two other linked markers, hem and phe, differs.     

Subcellular localization of the leucine biosynthetic enzymes in yeast

When baker's yeast spheroplasts were lysed by mild osmotic shock, practically all of the isopropylmalate isomerase and the beta-isopropylmalate dehydrogenase was released into the 30,000 x g supernatant fraction, as was the cytosol marker enzyme, glucose-6-phosphate dehydrogenase. alpha-Isopropylmalate synthase, however, was not detected in the initial supernatant, but could be progressively solubilized by homogenization, appearing more slowly than citrate synthase but faster than cytochrome oxidase. Of the total glutamate-alpha-ketoisocaproate transaminase activity, approximately 20% was in the initial soluble fraction, whereas solubilization of the remainder again required homogenization of the spheroplast lysate. Results from sucrose density gradient centrifugation of a cell-free particulate fraction and comparison with marker enzymes suggested that alpha-isopropylmalate synthase was located in the mitochondria. It thus appears that, in yeast, the first specific enzyme in the leucine biosynthetic pathway (alpha-isopropylmalate synthase) is particulate, whereas the next two enzymes in the pathway (isopropylmalate isomerase and beta-isopropylmalate dehydrogenase) are "soluble," with glutamate-alpha-ketoisocaproate transaminase activity being located in both the cytosol and particulate cell fractions.     

Enzymology and evolution of the pyruvate pathway to 2-oxobutyrate in Methanocaldococcus jannaschii

The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than to previously characterized isopropylmalate isomerase or homoaconitase enzyme genes. To identify the functions of these homologs, the four combinations of subunits were heterologously expressed in Escherichia coli, purified, and reconstituted to generate the iron-sulfur center of the holoenzyme. Only the combination of MJ0499 and MJ1277 proteins catalyzed isopropylmalate and citramalate isomerization reactions. This pair also catalyzed hydration half-reactions using citraconate and maleate. Another broad-specificity enzyme, isopropylmalate dehydrogenase (MJ0720), catalyzed the oxidative decarboxylation of beta-isopropylmalate, beta-methylmalate, and d-malate. Combined with these results, phylogenetic analysis suggests that the pyruvate pathway to 2-oxobutyrate (an alternative to threonine dehydratase in isoleucine biosynthesis) evolved several times in bacteria and archaea. The enzymes in the isopropylmalate pathway of leucine biosynthesis facilitated the evolution of 2-oxobutyrate biosynthesis through the introduction of a citramalate synthase, either by gene recruitment or gene duplication and functional divergence.     

The Product of the LEU-3 Cistron as a Regulatory Element for the Production of the Leucine Biosynthetic Enzymes of Neurospora

A class of intracistronic (or closely linked) partial reversions of leu-3 mutations has been found to be conditionally constitutive with respect to the synthesis of isopropylmalate isomerase (specified by the leu-2 cistron) and beta-isopropylmalate dehydrogenase (specified by the leu-1 cistron), two of the enzymes of leucine biosynthesis in Neurospora. The intermediate level of enzyme production by these leu-3(cc) mutants is independent of the obligatory inducer effector, alpha-isopropylmalate, but dependent upon the presence of the branched-chain amino acids, isoleucine, valine and leucine. The properties of leu-3+, leu-3 and leu-3(cc) in heterokaryons indicate that the transnuclear regulatory activity of the leu-3 product varies specifically as a function of available effector molecules. The information presented suggests that the leu-3 cistron is responsible not only for the production of a "positive" regulatory substance necessary for the expression of the leu-1 and leu-2 cistrons, but that it probably serves also a coordinating role in the expression of many of the genes involved in branched-chain amino acid metabolism.     

Regulation of synthesis of the branched-chain amino acids and cognate aminoacyl-transfer ribonucleic acid synthetases of Escherichia coli: a common regulatory element

Regulation of isoleucine, valine, and leucine biosynthesis and isoleucyl-, valyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined in two mutant strains of Escherichia coli. One mutant was selected for growth resistance to the isoleucine analogue, ketomycin, and the other was selected for growth resistance to both trifluoroleucine and valine. Control of the synthesis of the branched-chain amino acids by repression was altered in both of these mutants. They also exhibited altered control of formation of isoleucyl-tRNA synthetase (EC 6.1.15, isoleucine:sRNA ligase, AMP), valyl-tRNA synthetase (EC 6.1.1.9, valine:sRNA ligase, AMP), and leucyl-tRNA synthetase (EC 6.1.1.4, leucine:sRNA ligase, AMP). These results suggest the existence of a common element for the control of these two classes of enzymes in Escherichia coli.     

Characterization of Leucine Auxotrophs of the White Rot Basidiomycete Phanerochaete chrysosporium

Six leucine auxotrophic strains of the white rot basidiomycete Phanerochaete chrysosporium were characterized genetically and biochemically. Complementation studies involving the use of heterokaryons identified three leucine complementation groups. Since all of the leucine auxotrophs grew on minimal medium supplemented with α-ketoisocaproate as well as with leucine, the transaminase catalyzing the last step in the leucine pathway was apparently normal in all strains. Therefore, the wild-type, auxotrophic, and several heterokaryotic strains were assayed for the activities of the other enzymes specific to leucine biosynthesis. Leu2 and Leu4 strains (complementation group I) lacked only α-isopropylmalate synthase activity; Leu3 and Leu6 strains (group III) lacked isopropylmalate isomerase activity; and Leu1 and Leu5 strains (group II) lacked β-isopropylmalate dehydrogenase. Heterokaryons formed from leucine auxotrophs of different complementation groups had levels of activity for all three enzymes similar to those found in the wild-type strain.     

Transposed LEU2 gene of Saccharomyces cerevisiae is regulated normally.

The repression of beta-isopropylmalate dehydrogenase, the LEU2 gene product, by leucine and leucine plus threonine was unaffected by the transposition of LEU2 from its original locus on chromosome III to a new locus within the ribosomal deoxyribonucleic acid gene cluster on chromosome XII. Since the expression of the LEU2 gene is probably controlled at a pretranslational level, we conclude that the recombinant plasmid used for transformation carries regulatory information in addition to LEU2 structural information.     

Interferon suppresses steroid-inducible glutamine synthetase biosynthesis in embryonic chick neural retina.

Effect of chick interferon on the biosynthesis of glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) was studied in the embryonic chick neural retina cultures induced for the enzyme activity by hydrocortisone. The retinal enzyme radioactively labelled with [3H]leucine was precipitated by specific antibody against the enzyme isolated from adult chick liver. The immunological determination offered evidence that the suppressive effect of interferon on the hormonal induction of the enzyme was primarily due to reduced rate of its synthesis and accumulation.     

Interferon suppresses steroid-inducible glutamine synthetase biosynthesis in embryonic chick neural retina

Effect of chick interferon on the biosynthesis of glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) was studied in the embryonic chick neural retina cultures induced for the enzyme activity by hydrocortisone. The retinal enzyme radioactively labelled with [³H]leucine was precipitated by specific antibody against the enzyme isolated from adult chick liver. The immunological determination offered evidence that the suppressive effect of interferon on the hormonal induction of the enzyme was primarily due to reduced rate of its synthesis and accumulation.     

Cloning, Disruption and Chromosomal Mapping of Yeast LEU3 , a Putative Regulatory Gene

The LEU3 gene of the yeast Saccharomyces cerevisiae, which is involved in the regulation of at least two LEU structural genes (LEU1 and LEU2), has been cloned by complementation of leu3 mutations and shown to reside within a 5.6-kb fragment. Transformation of leu3 mutants with LEU3-carrying multicopy plasmids restored normal, leucine-independent growth behavior in the recipients. It also restored approximately wild-type levels of isopropylmalate isomerase (LEU1) and beta-isopropylmalate dehydrogenase (LEU2), which were strongly reduced when exogenous leucine was supplied. Strains containing a disrupted leu3 allele were constructed by deleting 0.7-kb of LEU3 DNA and inserting the yeast HIS3 gene in its place. Like other leu3 mutants, these strains were leaky leucine auxotrophs, owing to a basal level of expression of LEU1 and LEU2. Southern transfer and genetic analyses of strains carrying a disrupted leu3 allele demonstrated that the cloned gene was LEU3, as opposed to a suppressor. Disruption of LEU3 was performed also with a diploid and shown to be nonlethal by tetrad analysis. Northern transfer experiments showed that the LEU3 gene produces mRNA approximately 2.9 kilonucleotides in length. The leu3 marker was mapped to chromosome XII by the spo11 method. Linkage to ura4 by about 44 centiMorgans places leu3 on the right arm of this chromosome.     

Malic enzyme activity is not the only bottleneck for lipid accumulation in the oleaginous fungus Mucor circinelloides

Commercial interest in microbial lipids is increasing due to their potential use as feedstock for biodiesel production. The supply of NADPH generated by malic enzyme (ME; NADP⁺-dependent; EC 1.1.1.40) has been postulated as being the rate-limiting step for fatty acid biosynthesis in oleaginous fungi, based mainly on data from the zygomycete Mucor circinelloides studies. This fungus contains five genes that code for six different ME isoforms. One of these genes, malA, codes for the isoforms III and IV, which have previously been associated with lipid accumulation. Following a strategy of targeted integration of an engineered malA gene, a stable strain overexpressing malA and showing high ME activity has been obtained, demonstrating the feasibility of this strategy to overexpress genes of biotechnological interest in M. circinelloides. This is the first report showing the integration and overexpression of a gene in Zygomycetes. Unexpectedly, the genetically modified strain showed a lipid content similar to that of a prototrophic non-overexpressing control strain, suggesting that another limiting step in the fatty acid synthesis pathway may have been revealed as a consequence of the elimination of malic enzyme-based bottleneck. Otherwise, the fact that prototrophic strains showed at least a 2.5-fold increase in lipid accumulation in comparison with leucine auxotrophic strains suggests that a wild-type leucine biosynthetic pathway is required for lipid accumulation. Moreover, increasing concentrations of leucine in culture medium increased growth of auxotrophs but failed to increase lipid content, suggesting that the leucine synthesized by the fungus is the only leucine available for lipid biosynthesis. These results support previous data postulating leucine metabolism as one of the pathways involved in the generation of the acetyl-CoA required for fatty acid biosynthesis.