The primordial metabolism: an ancestral interconnection between leucine,arginine, and lysine biosynthesis

Background

It is generally assumed that primordial cells had small genomes with simple genes coding for enzymes able to react with a wide range of chemically related substrates, interconnecting different metabolic routes. New genes coding for enzymes with a narrowed substrate specificity arose by paralogous duplication(s) of ancestral ones and evolutionary divergence. In this way new metabolic pathways were built up by primordial cells. Useful hints to disclose the origin and evolution of ancestral metabolic routes and their interconnections can be obtained by comparing sequences of enzymes involved in the same or different metabolic routes. From this viewpoint, the lysine, arginine, and leucine biosynthetic routes represent very interesting study-models. Some of the lys, arg and leu genes are paralogs; this led to the suggestion that their ancestor genes might interconnect the three pathways. The aim of this work was to trace the evolutionary pathway leading to the appearance of the extant biosynthetic routes and to try to disclose the interrelationships existing between them and other pathways in the early stages of cellular evolution.

Results

The comparative analysis of the genes involved in the biosynthesis of lysine, leucine, and arginine, their phylogenetic distribution and analysis revealed that the extant metabolic "grids" and their interrelationships might be the outcome of a cascade of duplication of ancestral genes that, according to the patchwork hypothesis, coded for unspecific enzymes able to react with a wide range of substrates. These genes belonged to a single common pathway in which the three biosynthetic routes were highly interconnected between them and also to methionine, threonine, and cell wall biosynthesis. A possible evolutionary model leading to the extant metabolic scenarios was also depicted.

Conclusion

The whole body of data obtained in this work suggests that primordial cells synthesized leucine, lysine, and arginine through a single common metabolic pathway, whose genes underwent a set of duplication events, most of which can have predated the appearance of the last common universal ancestor of the three cell domains (Archaea, Bacteria, and Eucaryotes). The model proposes a relative timing for the appearance of the three routes and also suggests a possible evolutionary pathway for the assembly of bacterial cell-wall.

     

Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus

Alpha-aminoadipate aminotransferase (AAA-AT), a homolog of mammalian kynurenine aminotransferase II (Kat II), transfers an amino group to 2-oxoadipate to yield alpha-aminoadipate in lysine biosynthesis through the alpha-aminoadipate pathway in Thermus thermophilus. AAA-AT catalyzes transamination against various substrates, including AAA, glutamate, leucine, and aromatic amino acids. To elucidate the structural change for recognition of various substrates, we determined crystal structures of AAA-AT in four forms: with pyridoxal 5'-phosphate (PLP) (PLP complex), with PLP and leucine (PLP/Leu complex), with N-phosphopyridoxyl-leucine (PPL) (PPL complex), and with N-phosphopyridoxyl-alpha-aminoadipate (PPA) at 2.67, 2.26, 1.75, and 1.67 A resolution, respectively. The PLP complex is in an open state, whereas PLP/Leu, PPL, and PPA complexes are in closed states with maximal displacement (over 7 A) of the alpha2 helix and the beta1 strand in the small domain to cover the active site, indicating that conformational change is induced by substrate binding. In PPL and PLP/Leu complexes, several hydrophobic residues on the alpha2 helix recognize the hydrophobic side chain of the bound leucine moiety whereas, in the PPA complex, the alpha2 helix rotates to place the guanidium moiety of Arg23 on the helix at the appropriate position to interact with the carboxyl side chain of the AAA moiety. These results indicate that AAA-AT can recognize various kinds of substrates using the mobile alpha2 helix. The crystal structures and site-directed mutagenesis revealed that intersubunit-electrostatic interactions contribute to the elevated thermostability of this enzyme.     

Horizontally acquired DAP pathway as a unit of self-regulation

In this study, we report the acquisition of the diaminopimelic acid (DAP) pathway of lysine biosynthesis in choanoflagellate Monosiga brevicollis and investigate how this pathway is incorporated and regulated in the established metabolic network. Our data show that all major genes related to the DAP pathway in Monosiga were acquired from bacteria and algae. Although an endogenous lysC exists in Monosiga, the newly acquired lysC is fused to lysA and used specifically for lysine biosynthesis. In addition, these acquired genes encode two key rate-limiting enzymes, thus conferring Monosiga a self-regulated unit with ability to generate lysine. Our data suggest that a newly acquired metabolic capability can be added to the recipient organism without disturbing the previously established metabolic network. This finding also implies that the biochemical system of the recipient organism may determine the type and function of genes to be acquired.     

Characterization of homoisocitrate dehydrogenase involved in lysine biosynthesis of an extremely thermophilic bacterium,Thermus thermophilus HB27, and evolutionary implication of beta-decarboxylating dehydrogenase

Although the presence of an enzyme that catalyzes beta-decarboxylating dehydrogenation of homoisocitrate to synthesize 2-oxoadipate has been postulated in the lysine biosynthesis pathway through alpha-aminoadipate (AAA), the enzyme has not yet been analyzed at all, because no gene encoding the enzyme has been identified until recently. A gene encoding a protein with a significant amino acid sequence identity to both isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase was cloned from Thermus thermophilus HB27. The gene product produced in recombinant Escherichia coli cells demonstrated homoisocitrate dehydrogenase (HICDH) activity. A knockout mutant of the gene showed an AAA-auxotrophic phenotype, indicating that the gene product is involved in lysine biosynthesis through AAA. We therefore named this gene hicdh. HICDH, the gene product, did not catalyze the conversion of 3-isopropylmalate to 2-oxoisocaproate, a leucine biosynthetic reaction, but it did recognize isocitrate, a related compound in the tricarboxylic acid cycle, as well as homoisocitrate as a substrate. It is of interest that HICDH catalyzes the reaction with isocitrate about 20 times more efficiently than the reaction with the putative native substrate, homoisocitrate. The broad specificity and possible dual function suggest that this enzyme represents a key link in the evolution of the pathways utilizing citrate derivatives. Site-directed mutagenesis study reveals that replacement of Arg(85) with Val in HICDH causes complete loss of activity with isocitrate but significant activity with 3-isopropylmalate and retains activity with homoisocitrate. These results indicate that Arg(85) is a key residue for both substrate specificity and evolution of beta-decarboxylating dehydrogenases.     

Genetic characterization of a highly efficient alternate pathway of serine biosynthesis in Escherichia coli.

There exists in Escherichia coli a known set of enzymes that were shown to function in an efficient and concerted way to convert threonine to serine. The sequence of reactions catalyzed by these enzymes is designated the Tut cycle (threonine utilization). To demonstrate that the relevant genes and their protein products play essential roles in serine biosynthesis, a number of mutants were analyzed. Strains of E. coli with lesions in serA, serB, serC, or glyA grew readily on minimal medium supplemented with elevated levels of leucine, arginine, lysine, threonine, and methionine. No growth on this medium was observed upon testing double mutants with lesions in one of the known ser genes plus a second lesion in glyA (serine hydroxymethyltransferase), gcv (the glycine cleavage system), or tdh (threonine dehydrogenase). Pseudorevertants of ser mutants capable of growth on either unsupplemented minimal medium or medium supplemented with low levels of leucine, arginine, lysine, threonine, and methionine were isolated. At least two unlinked mutations were associated with such phenotypes.     

Patterns of Methionine and Lysine Biosynthesis in the Trypanosomatidae During Growth*

SYNOPSIS. Nine Crithidia spp., 2 Blastocrithidia spp., 3 Leptomonas spp. and 2 Trypanosoma spp. were tested for ability to synthesize methionine and lysine during growth. A prerequisite for methionine biosynthesis is an inordinately high level of folic acid (0.1 mg/100 ml) in the medium. Crithidia factor-type unconjugated pteridines cannot spare this requirement. Since the methionine-synthesis factor is still present after acid hydrolysis which destroys folic acid, the factor is either a breakdown product of folic acid or an impurity in the commercial product. All save C. fasciculata var. noelleri and C. from Syrphid could synthesize methionine from homocysteine thiolactone. None of the organisms synthesized lysine from α-aminoadipic acid (AAA), thus ruling out the existence of the AAA pathway for lysine synthesis in the Trypanosomatidae. Nine of the organisms synthesized lysine from a mixture of LL- and meso-α,e-diaminopimelic acid. Since both LL- and meso-DAP are intermediates in the biosynthesis of lysine by the DAP pathway (LL-DAP→meso-DAP→lysine) and since decarboxylation of either LL- or meso-DAP could result in formation of lysine, pure meso-DAP was tested and found active. Thus at least the terminal portion of the DAP pathway for lysine synthesis exists in these true animal cells. Statements about absence of ability to synthesize lysine in animal cells and consequent evolutionary interpretations will therefore require revision.     

Biosynthesis of lysine in plants: the putative role of meso-diaminopimelate dehydrogenase

Extracts from Chlamydomonas, corn, soybean and tobacco were tested for enzymes of the lysine biosynthetic pathway. Dihydrodipicolinic acid (DHD) synthase, DHD reductase, diaminopimelate (DAP) epimerase and DAP decarboxylase were present in all. However, in contrast to the report of Wenko et al., meso-DAP dehydrogenase could not be detected in extracts prepared from soybean. Moreover, it was not found in Chlamydomonas, corn and tobacco as well. In order to set an upper limit to the amount of meso-DAP dehydrogenase that might be present, reconstruction experiments were performed with soybean and corn extracts in which the conversion of dihydrodipicolinate to lysine was made dependent on the addition of limited amounts of the meso-DAP dehydrogenase purified from Bacillus sphaericus. The presence of DAP epimerase and the absence of meso-DAP dehydrogenase indicates that the meso-DAP dehydrogenase abbreviated pathway for lysine synthesis is not operative in plants.     

Biochemical and phylogenetic characterization of a novel diaminopimelate biosynthesis pathway in prokaryotes identifies a diverged form of LL-diaminopimelate aminotransferase

A variant of the diaminopimelate (DAP)-lysine biosynthesis pathway uses an LL-DAP aminotransferase (DapL, EC 2.6.1.83) to catalyze the direct conversion of L-2,3,4,5-tetrahydrodipicolinate to LL-DAP. Comparative genomic analysis and experimental verification of DapL candidates revealed the existence of two diverged forms of DapL (DapL1 and DapL2). DapL orthologs were identified in eubacteria and archaea. In some species the corresponding dapL gene was found to lie in genomic contiguity with other dap genes, suggestive of a polycistronic structure. The DapL candidate enzymes were found to cluster into two classes sharing approximately 30% amino acid identity. The function of selected enzymes from each class was studied. Both classes were able to functionally complement Escherichia coli dapD and dapE mutants and to catalyze LL-DAP transamination, providing functional evidence for a role in DAP/lysine biosynthesis. In all cases the occurrence of dapL in a species correlated with the absence of genes for dapD and dapE representing the acyl DAP pathway variants, and only in a few cases was dapL coincident with ddh encoding meso-DAP dehydrogenase. The results indicate that the DapL pathway is restricted to specific lineages of eubacteria including the Cyanobacteria, Desulfuromonadales, Firmicutes, Bacteroidetes, Chlamydiae, Spirochaeta, and Chloroflexi and two archaeal groups, the Methanobacteriaceae and Archaeoglobaceae.     

The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa

The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.     

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.     

Biosynthesis of lysine in plants: evidence for a variant of the known bacterial pathways

With the aim of elucidating how plants synthesize lysine, extracts prepared from corn, tobacco, Chlamydomonas and soybean were tested and found to lack detectable amounts of N-alpha-acyl-L,L-diaminopimelate deacylase or N-succinyl-alpha-amino-epsilon-ketopimelate-glutamate aminotransaminase, two key enzymes in the central part of the bacterial pathway for lysine biosynthesis. Corn extracts missing two key enzymes still carried out the overall synthesis of lysine when provided with dihydrodipicolinate. An analysis of available plant DNA sequences was performed to test the veracity of the negative biochemical findings. Orthologs of dihydrodipicolinate reductase and diaminopimelate epimerase (enzymes on each side of the central pathway) were readily found in the Arabidopsis thaliana genome. Orthologs of the known enzymes needed to convert tetrahydrodipicolinate to diaminopimelic acid (DAP) were not detected in Arabidopsis or in the plant DNA sequence databases. The biochemical and reinforcing bioinformatics results provide evidence that plants may use a novel variant of the bacterial pathways for lysine biosynthesis.     

L-arginine utilization by Pseudomonas species

The utilization of arginine was studied in several different Pseudomonas species. The arginine decarboxylase and agmatine deiminase pathways were found to be characteristic of Pseudomonas species of group I as defined by Palleroni et al. (1974). Pseudomonas putida strains had three distinct arginine catabolic pathways initiated by arginine decarboxylase, arginine deiminase and arginine oxidase, respectively. The two former routes were also present in P. fluorescens and P. mendocina and in P. aeruginosa which also used arginine by a further unknown pathway. None of these pathways occurred in P. cepacia strains; agmatine catabolism seemed to follow an unusual route involving guanidinobutyrate as intermediate.     

Regulation of lysine and dipicolinic acid biosynthesis in Bacillus brevis ATCC 10068: significance of derepression of the enzymes during the change from vegetative growth to sporulation

Lysine biosynthetic pathway enzymes of Bacillus brevis ATCC 1068 were studied as a function of stage of development (growth and sporulation). The synthesis of aspartic-2-eemialdehyde dehydrogenase (ASA-dehydrogenase), dihydrodipicolinate synthase (DHDPA-synthase), DHPA-reductase and diaminopimelate decarboxylase (DAP-decarboxylase) was found not to be co-regulated, since lysine was not a co-repressor for these enzymes. Unlike the aspartokinase isoenzymes, the other enzymes of the lysine pathway were not derepressed in thiosine-resistant, lysine-excreting mutants. Thus, the aspartokinase isoenzymes were the key enzymes during growth and regulation of lysine biosynthesis through restriction of l-ASA synthesis via feedback control by lysine on the aspartokinases was therefore suggested.In contrast to other Bacillus species, the levels of the lysine biosynthetic pathway enzymes of strain ATCC 10068 were not derepressed during the change from vegetative growth to sporulation. Two control mechanisms, enabling the observed preferential channelling of carbon for the synthesis of spore-specific diaminopimelic acid (DAP) and dipicolinic acid (DPA) were a) loss of DAP-decarboxylase, b) inhibition of DHDPA-reductase by DPA. Increase in the level of the DAP pool during sporulation, as a consequence of the loss of DAP-decarboxylase, and its relevance to the non-enzymatic formation of DPA has been discussed.Abbreviations l-ASA l-aspartic-2-semialdehyde - DAP diaminopimelic acid - DPA dipicolinic acid - DHDPA dihydrodipicolinate - AGM aspargine-glycerol medium - PY peptone-yeast extract - NB+NSM nutrient broth plus nutrient sporulation medium     

Development of stable, genetically well-defined conditionally viableEscherichia coli strains

Summary The diaminopimelate (DAP) pathway provides the cell with lysine and with DAP, a vital cell wall constituent. Mutations in the DAP pathway of lysine biosynthesis are lethal for cells exposed to lysine in the absence of DAP. In this paper, the substitution of thedapD gene ofEscherichia coli with the kanamycin resistance gene from Tn903 is described and its possible uses are discussed.     

Efficient biosynthesis of α-aminoadipic acid via lysine catabolism in Escherichia coli

α-Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high-level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine-α-ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α-aminoadipate-δ-semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production.     

Cloning and characterization of argR, a gene that participates in regulation of arginine biosynthesis and catabolism in Pseudomonas aeruginosa PAO1.

Gel retardation experiments indicated the presence in Pseudomonas aeruginosa cell extracts of an arginine-inducible DNA-binding protein that interacts with the control regions for the car and argF operons, encoding carbamoylphosphate synthetase and anabolic ornithine carbamoyltransferase, respectively. Both enzymes are required for arginine biosynthesis. The use of a combination of transposon mutagenesis and arginine hydroxamate selection led to the isolation of a regulatory mutant that was impaired in the formation of the DNA-binding protein and in which the expression of an argF::lacZ fusion was not controlled by arginine. Experiments with various subclones led to the conclusion that the insertion affected the expression of an arginine regulatory gene, argR, that encodes a polypeptide with significant homology to the AraC/XylS family of regulatory proteins. Determination of the nucleotide sequence of the flanking regions showed that argR is the sixth and terminal gene of an operon for transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Furthermore, argR inactivation abolished the induction of several enzymes of the arginine succinyltransferase pathway, which is considered the major route for arginine catabolism under aerobic conditions. Consistent with this finding and unlike the parent strain, the argR::Gm derivative was unable to utilize arginine or ornithine as the sole carbon source. The combined data indicate a major role for ArgR in the control of arginine biosynthesis and aerobic catabolism.     

Regulation of lysine biosynthesis in Escherichia coli K12.

A general survey of the regulation in lysine biosynthesis in Escherichia coli K12 is presented. No polygenic operon exists for the genes that code for enzymes of the lysine biosynthetic pathway. Lysyl-tRNA is not directly involved as a co-repressor in the pathway. Different regulation mechanisms must exist for the different enzymes. In the case of the last enzyme, diaminopimelate decarboxylase, its synthesis is induced in vivo by the lysine-sensitive aspartokinase under its non-inhibited allosteric conformation.     

Regulation of lysine catabolism in higher plants

Lysine is an essential amino acid for mammals but its concentration in cereals, one of our main food sources, is low. Research over the past 40 years has unraveled many biochemical and molecular details of the aspartic acid pathway, which is the main route of lysine biosynthesis in plants. However, genetic manipulation of this pathway has not been successful at producing high-lysine seeds. This is because lysine, instead of being accumulated, is degraded via the saccharopine pathway. Recent work has increased our knowledge of this pathway, including both the enzymes involved and their regulation.