Functional and evolutionary relationship between arginine biosynthesis and prokaryotic lysine biosynthesis through alpha-aminoadipate

Our previous studies revealed that lysine is synthesized through alpha-aminoadipate in an extremely thermophilic bacterium, Thermus thermophilus HB27. Sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from alpha-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis. In the present study, we cloned an argD homolog of T. thermophilus HB27 which was not included in the previously cloned lysine biosynthetic gene cluster and determined the nucleotide sequence. A knockout of the argD-like gene, now termed lysJ, in T. thermophilus HB27 showed that this gene is essential for lysine biosynthesis in this bacterium. The lysJ gene was cloned into a plasmid and overexpressed in Escherichia coli, and the LysJ protein was purified to homogeneity. When the catalytic activity of LysJ was analyzed in a reverse reaction in the putative pathway, LysJ was found to transfer the epsilon-amino group of N(2)-acetyllysine, a putative intermediate in lysine biosynthesis, to 2-oxoglutarate. When N(2)-acetylornithine, a substrate for arginine biosynthesis, was used as the substrate for the reaction, LysJ transferred the delta-amino group of N(2)-acetylornithine to 2-oxoglutarate 16 times more efficiently than when N(2)-acetyllysine was the amino donor. All these results suggest that lysine biosynthesis in T. thermophilus HB27 is functionally and evolutionarily related to arginine biosynthesis.     

Cloning and organization of seven arginine biosynthesis genes from Neisseria gonorrhoeae.

A genomic library for Neisseria gonorrhoeae, constructed in the lambda cloning vector EMBL4, was screened for clones carrying arginine biosynthesis genes by complementation of Escherichia coli mutants. Clones complementing defects in argA, argB, argE, argG, argIF, carA, and carB were isolated. An E. coli defective in the acetylornithine deacetylase gene (argE) was complemented by the ornithine acetyltransferase gene (argJ) from N. gonorrhoeae. This heterologous complementation is reported for the first time. The carAB operon from E. coli hybridized with the gonococcal clones that carried carA or carB genes under conditions of high stringency, detecting 80% or greater similarity and showing that the nucleotide sequence of the carbamoylphosphate synthetase genes is very similar in these two organisms. Under these conditions for hybridization, the gonococcal clones carrying argB or argF genes did not hybridize with plasmids containing the corresponding E. coli gene. Cocomplementation experiments established gene linkage between carA and carB. Clones complementing a gene defect in argE were also able to complement an argA mutation. This suggests that the enzyme ornithine acetyltransferase from N. gonorrhoeae (encoded by argJ) may be able to complement both argA and argE mutations in E. coli. The arginine biosynthesis genes in N. gonorrhoeae appear to be scattered as in members of the family Pseudomonadaceae.     

Acetylornithine deacetylase, succinyldiaminopimelate desuccinylase and carboxypeptidase G2 are evolutionarily related.

The nucleotide (nt) sequence of the Escherichia coli argE gene, encoding the acetylornithine deacetylase (AO) subunit, has been established and corresponds to a 43-kDa (M(r) 42,320) polypeptide. The enzyme has been purified to near homogeneity and it appears to be a dimer consisting of two 43-kDa subunits. The amino acid sequence deduced from the nt sequence was compared to that of the subunit of E. coli succinyldiaminopimelate desuccinylase (the dapE gene product involved in the diaminopimelate pathway for lysine biosynthesis), since both enzymes share functional and biochemical features. Significant similarity covering the entire sequence allows us to infer a common origin for both deacylases. This homology extends to the Pseudomonas sp. G2 carboxypeptidase (G2CP); this or a functionally related enzyme may be responsible for the minor AO activity found in organisms relying on ornithine acetyltransferase for ornithine biosynthesis.     

Molecular Evolution of the Lysine Biosynthetic Pathways

Among the different biosynthetic pathways found in extant organisms, lysine biosynthesis is peculiar because it has two different anabolic routes. One is the diaminopimelic acid pathway (DAP), and the other over the a-aminoadipic acid route (AAA). A variant of the AAA route that includes some enzymes involved in arginine and leucine biosyntheses has been recently reported in Thermus thermophilus (Nishida et al. 1999). Here we describe the results of a detailed genomic analysis of each of the sequences involved in the two lysine anabolic routes, as well as of genes from other routes related to them. No evidence was found of an evolutionary relationship between the DAP and AAA enzymes. Our results suggest that the DAP pathway is related to arginine metabolism, since the lysC, asd, dapC, dapE, and lysA genes from lysine biosynthesis are related to the argB, argC, argD, argE, and speAC genes, respectively, whose products catalyze different steps in arginine metabolism. This work supports previous reports on the relationship between AAA gene products and some enzymes involved in leucine biosynthesis and the tricarboxylic acid cycle (Irvin and Bhattacharjee 1998; Miyazaki et al. 2001). Here we discuss the significance of the recent finding that several genes involved in the arginine (Arg) and leucine (Leu) biosynthesis participate in a new alternative route of the AAA pathway (Miyazaki et al. 2001). Our results demonstrate a clear relationship between the DAP and Arg routes, and between the AAA and Leu pathways.     

N6-(1-carboxyethyl)lysine formation by Streptococcus lactis. Purification, synthesis, and stereochemical structure

During growth in an arginine-deficient (chemically defined) medium, cells of Streptococcus lactis K1 formed significant amounts of a previously undetected ninhydrin-positive compound. This intracellular compound did not cochromatograph with any of a wide range of amino acids or amino acid analogs tested. However, by two-dimensional thin layer chromatography, the unknown compound migrated close to the recently discovered N5-(1-carboxyethyl)ornithine (Thompson, J., Curtis, M. A., and Miller, S. P. F. (1986) J. Bacteriol. 167, 522-529; Miller, S. P. F., and Thompson, J. (1987) J. Biol. Chem. 262, 16109-16115). The purified compound behaved as a neutral amino acid and eluted between valine and methionine in the amino acid analyzer. The results of 1H NMR spectroscopy suggested the presence of a lysine backbone and a coupled methyl-methine unit in the molecule, and 13C NMR showed that there were nine carbon atoms, of which two (C-1 and C-7) were carboxyl carbons. The simplest structure compatible with the physicochemical data was that of an alkylated derivative of lysine. The identity of this new amino acid, N6-(1-carboxyethyl)lysine, was confirmed by chemical synthesis. In vivo labeling experiments conducted using L[U-14C]lysine and [epsilon-15N]lysine showed that exogenous lysine served as the precursor of intracellular N6-(1-carboxyethyl)lysine and that the epsilon-amino N atom was conserved during biosynthesis of the lysine derivative. Of the two possible diastereomers (2S,8S or 2S,8R) of N6-(1-carboxyethyl)lysine, comparative 13C NMR spectroscopy established that the amino acid produced by S. lactis K1 was exclusively of the 2S,8S configuration.     

The regulatory region of the divergent argECBH operon in Escherichia coli K-12

The nucleotide sequence of the control region of the divergent argECBH operon has been established in the wild type and in mutants affecting expression of these genes. The argE and argCBH promoters face each other and overlap with an operator region containing two domains which may act as distinct repressor binding sites. A long leader sequence - not involved in attenuation - precedes argCBH. Overlapping of the argCBH promoter and the region involved in ribosome mobilization for argE translation explains the dual effect of some mutations. Mutations causing semi-constitutive expression of argE improve putative promoter sequences within argC. Implications of these results regarding control mechanisms in amino acid biosynthesis and their evolution are discussed.     

Evidence for non-enzymatic glycosylation of Escherichia coli chromosomal DNA

We have recently shown that the process of non-enzymatic glycosylation (glycation) takes place in Escherichia coli under physiological conditions and affects both recombinant and endogenous bacterial proteins. In this study, we further demonstrate that E. coli chromosomal DNA is also subjected to glycation under physiological growth conditions. The E. coli DNA accumulates early glycation (Amadori) products as proven by the nitroblue tetrazolium (NBT) reduction assay. It showed also immunoreactivity to a monoclonal antibody raised against N(in)-(carboxymethyl)lysine and fluorescent properties indicative of modifications with advanced glycation end-products. Two types of fluorophores were detected in the E. coli DNA with excitation maxima at 360 nm and 380 nm and emission maxima at 440 nm and 410 nm. Using the NBT reduction assay, fluorescence spectroscopy and enzyme-linked immunosorbent assay we revealed that glycation adducts accumulate in DNA predominantly in the stationary phase of growth, although they could be detected also in exponential-phase cells. Besides on the growth phase, the extent of DNA glycation depends also on the nutrient broth composition being more extensive in rich media. Thiamine was found to inhibit both DNA glycation and spontaneous point mutations as judged by the decreased rate of the argE3 to Arg(+) reversions in the E. coli strain AB1157.     

Arginine biosynthesis in Campylobacter jejuni TGH9011: determination of the argCOBD cluster

Arginine biosynthetic genes from Campylobacter jejuni TGH9011 were cloned by functional complementation of the respective Escherichia coli arginine biosynthetic mutants. Complementation of argA, argB, argC, argD, argE, argF, and argH auxotrophs was accomplished using a pBR322-based C. jejuni TGH9011 plasmid library. By cross-complementation analyses, the first four steps of arginine biosynthesis were shown to be closely linked on the genome. Two additional clones complementing the first (ArgA) and fifth (ArgE) steps in arginine biosynthesis were obtained. Neither recombinant showed linkage to the arg cluster, to each other, nor to other arginine biosynthetic functions by cross-complementation. Genes argF and argH were not linked to other arginine biosynthetic genes by cross-complementation analysis. Restriction enzyme patterns of recombinant plasmids fell into five groups. Group I contained the arg(ABCD) complementing locus. Group II and Group III were the two genetic loci corresponding to the argA and argE complementing genes. Group II contains the hipO gene encoding N-benzoylglycine-amino-acid amidohydrolase, also known as hippurate hydrolase. Group III contains the hipO homolog of C. jejuni. Group IV represents the argF gene. Group V is the argH gene. Functional complementation of mutations in the first four steps of the arginine biosynthetic pathway was obtained on recombinant plasmid pARGC2. The predicted order of gene complementation was argCargA(argBargD). The sequence of the insert in plasmid pARGC2 revealed direct homologs for argC, argB, and argD. However, sequence analysis of the gene complementing ArgA function in two separate E. coli argA mutants determined that the C. jejuni gene was not a canonical argA gene. The gene complementing the argA defect, which we call argO, showed limited homology to the streptothricin acetyltransferase gene (sat) of Escherichia coli. The flanking open reading frames in pARGC2 showed no homologies to arginine biosynthetic genes. The structure of the argCOBD gene arrangement is discussed with reference to the presence and location of other arginine biosynthetic genes on the genome of C. jejuni and other bacterial organisms.     

Arginine and pyrimidine biosynthetic defects in Neisseria gonorrhoeae strains isolated from patients

Neisseria gonorrhoeae strains with nutritional requirements that include arginine (Arg-), uracil (Ura-), and hypoxanthine have attracted attention because of their tendency to cause disseminated infections, as a basis for genetic studies of arginine and pyrimidine biosynthesis, we examined the activities of four enzymes of these pathways in cell-free extracts of both prototrophic and Arg- Ura- strains. Activities of glutamate acetyltransferase, aspartate transcarbamylase, and orotate phosphoribosyltransferase, encoded respectively by argE, pyrB, and pyrE, were absent in some Arg- Ura- isolates. Gonococci that were unable to utilize ornithine for growth in place of citrulline lacked activity of carbamyl phosphate synthetase (encoded by car). Defects of car imposed requirements for both citrulline (or arginine) and a pyrimidine because of the dual role of carbamyl phosphate in the two pathways. Defects of argE, car, pyrB, and pyrE were separately introduced by genetic transformation into representatives of a gonococcal strain which initially was prototrophic. Results of enzyme assays of these isogenic auxotrophic transformants confirmed the gene-enzyme relationships.     

Turn-on of Inactive Genes by Promoter Recruitment in ESCHERICHIA COLI: Inverted Repeats Resulting in Artificial Divergent Operons

We have characterized two rearrangements consisting of inverted repeats of the argE gene. The promoters (p) of argE and of argCBH face each other over an internal operator. The rearrangements were obtained as reactivations of argE in a strain harboring an argEp deletion on a lambda darg prophage. In both cases the repeat included argE and argCBHp on either side of a unique sequence; the result is a divergent operon in which each copy of argCBHp reads into the adjacent argE repeat. In one case, the pair of repeats adjoins the silent parental gene, forming a triplication (comes from leads to comes from). The other rearrangement consists of a single argE palindrome, but the whole prophage is rearranged into an inverted repeat, analogous to certain lambda dv's. Both structures could be explained by breakage of a replication fork passing argE and by inaccurate rejoining of strands. The lambda dv-like rearrangement would result from breakage at both replication forks of a phage or prophage replicating during transient release of immunity. The triplication would imply breaking of a chromosomal replication fork, formation of a cyclic intermediate by recombination between the daughter duplex molecules and reinsertion into the parental argE gene. Formation of a triplication by replication errors involving appropriate strand switchings and branch migrations can not be excluded however.     

Regulatory mechanisms after short‐ and long‐term perturbed lysine biosynthesis in the aspartate pathway: the need for isogenes in Arabidopsis thaliana

The aspartate‐derived amino acid pathway in plants is an intensively studied metabolic pathway, because of the biosynthesis of the four essential amino acids lysine, threonine, isoleucine and methionine. The pathway is mainly controlled by the key regulatory enzymes aspartate kinase (AK; EC 2.7.2.4), homoserine dehydrogenase (HSDH; EC 1.1.1.3) and 4‐hydroxy‐tetrahydrodipicolinate synthase (EC 4.3.3.7), formerly referred to as dihydrodipicolinate synthase (DHDPS). They are encoded by isoenzyme families and it is not known why such families are evolutionarily maintained. To gain more insight into the specific roles and regulation of the isoenzymes, we inhibited DHDPS in Arabidopsis thaliana with the chemical compound (N,N‐dimethylglycinatoboranyloxycarbonylmethyl)‐dimethylamine‐borane (DDAB) and compared the short‐term effects on the biochemical and biomolecular level to the long‐term adaptations in dhdps knockout mutants. We found that DHDPS2 plays a crucial role in controlling lysine biosynthesis, thereby stabilizing flux through the whole aspartate pathway. Moreover, DHDPS2 was also shown to influence the threonine level to a large extent. In addition, the lysine‐sensitive AKs, AKLYS1 and AKLYS3 control the short‐ and long‐term responses to perturbed lysine biosynthesis in Arabidopsis thaliana.     

The yeast VAP homolog Scs2p has a phosphoinositide-binding ability that is correlated with its activity

The yeast VAMP-associated protein (VAP) homolog Scs2p is an endoplasmic reticulum (ER)/nuclear membrane protein that binds to an FFAT (diphenylalanine in an acidic tract) motif found in various lipid-metabolic proteins, including Opi1p, a negative regulator of phospholipid biosynthesis. Here, we show that Scs2p is a novel phosphoinositide-binding protein that can bind to phosphatidylinositol monophosphates and bisphosphates in vitro. The phosphoinositide-binding domain was assigned to the N-terminal major sperm protein (MSP) domain which also contains the FFAT-binding domain. When several lysine residues in the MSP domain were substituted for alanine, the resulting mutant Scs2 proteins lost the phosphoinositide-binding ability and failed to complement the inositol auxotrophy of an scs2 deletion strain. However, the mutant proteins still localized in the ER/nuclear membrane, in a similar manner to wild-type Scs2p. These results suggest the possibility that Scs2p activity is regulated by phosphoinositides to coordinate phospholipid biosynthesis in response to changes in phospholipid composition.     

Reversion of argE3 ochre strain Escherichia coli AB1157 as a tool for studying the stationary-phase (adaptive) mutations

Adaptive (starvation-associated) mutations occur in non-dividing cells and allow growth under the selective conditions imposed. We developed a new method for the determination of adaptive mutations in Escherichia coli. The system involves reversion to prototrophy of the argE3OC mutation and was tested on AB1157 strains mutated in the mutT and/or mutY genes. The bacteria that mutated adaptively grow into colonies on minimal medium plates devoid of arginine (starvation conditions) when incubated longer than 4 days. Using the replica plating method we solved the problem of discrimination between growth-dependent and adaptive argE3-->Arg+ revertants. Phenotype analysis and susceptibility of the Arg+ revertants to a set of T4 phage mutants create an additional possibility to draw a distinction between these two types of Arg+ revertants.     

The biosynthesis of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Alignment of the butylamine segment and source of the secondary amino nitrogen

The unusual amino acid hypusine is produced in a single protein of mammalian cells by a novel posttranslational event in which a lysine residue is conjugated with the four-carbon moiety from the polyamine spermidine to form an intermediate deoxyhypusine, and in which this intermediate is subsequently hydroxylated. Specifically isotopically labeled precursors of hypusine were used to identify the biosynthetic origin of some of the atoms of hypusine and thus to provide further insight into the mechanism of this in vivo chemical modification reaction. Radiolabel from [1,4-3H] putrescine, [1,8-3H]spermidine, and [5-3H]spermidine entered hypusine during growth of Chinese hamster ovary cells. The occurrence of this label at positions 1 and 4, at position 4, and at position 1, respectively, in the 4-amino-2-hydroxybutyl portion of hypusine revealed an alignment of atoms identical to that in the butylamine segment of spermidine. Growth of cells with [epsilon-15N]lysine as the source of lysine yielded hypusine enriched in 15N, whereas only isotope-free hypusine during growth by [4-15N]spermidine. These was found in cells whose spermidine was replaced during growth by [4-15N]spermidine. These findings are in accordance with a proposal that the first phase of hypusine biosynthesis, the production of intermediate deoxyhypusine, occurs through transfer of the butylamine moiety from spermidine to the epsilon-amino nitrogen of protein-bound lysine. The technique of thermospray high-performance liquid chromatography/mass spectrometry provided positive identification of 15N in hypusine through final separation and on-column direct analysis of this amino acid. Methods of preparation are given for spermidine of high specific radioactivity, labeled specifically at position 5 with 3H, and for spermidine with 15N at the 4-position.     

Dihydrodipicolinate reductase-like protein, CRR1, is essential for chloroplast NAD(P)H dehydrogenase in Arabidopsis

Chloroplast NAD(P)H dehydrogenase (NDH) is a homolog of the bacterial NADH dehydrogenase NDH-1 and is involved in cyclic electron transport around photosystem I. In higher plants, 14 subunits of the NDH complex have been identified. The subunit that contains the electron donor-binding site or an electron donor to NDH has not been determined. Arabidopsis crr1 (chlororespiratory reduction 1) mutants were isolated by chlorophyll fluorescence imaging on the basis of their lack of NDH activity. CRR1 is homologous to dihydrodipicolinate reductase (DHPR), which functions in a lysine biosynthesis pathway. However, the dihydrodipicolinate-binding motif was not conserved in CRR1, and the crr1 defect was specific to accumulation of the NDH complex, implying that CRR1 is not involved in lysine biosynthesis in Arabidopsis. Similarly to other nuclear-encoded genes for NDH subunits, CRR1 was expressed only in photosynthetic tissue. CRR1 contained a NAD(P)H-binding motif and was a candidate electron donor-binding subunit of the NDH complex. However, CRR1 was detected in the stroma but not in the thylakoid membranes, where the NDH complex is localized. Furthermore, CRR1 was stable in crr2-2 lacking the NDH complex. These results suggest that CRR1 is involved in biogenesis or stabilization of the NDH complex, possibly via the reduction of an unknown substrate.     

Sequence analysis and complementation studies of the argJ gene encoding ornithine acetyltransferase from Neisseria gonorrhoeae.

Clinical isolates of Neisseria gonorrhoeae frequently are deficient in arginine biosynthesis. These auxotrophs often have defects in the fifth step of the arginine biosynthetic pathway, the conversion of acetylornithine to ornithine. This reaction is catalyzed by the enzyme ornithine acetyltransferase, which is a product of the argJ gene. We have cloned and sequenced the gonococcal argJ gene and found that it contains an open reading frame of 1,218 nucleotides and encodes a peptide with a deduced Mr of 42,879. This predicted size was supported by minicell analysis. This gene was capable of complementing both Escherichia coli argE and argA mutations and of transforming an ArgJ- strain of N. gonorrhoeae to Arg+. Southern blots were able to detect bands that specifically hybridized to the gonococcal argJ gene in genomic DNA from Pseudomonas aeruginosa but not E. coli, a result that reflects the divergent nature of the arginine biosynthetic pathway in these organisms.     

The activity of a thermostable lipoyl synthase from Sulfolobus solfataricus with a synthetic octanoyl substrate

The protein lipoyl synthase (LipA) is essential for lipoic acid biosynthesis via sulfur insertions into a protein-bound octanoyl group. We have developed an in vitro assay for LipA using a synthetic tetrapeptide substrate, containing an N(epsilon)-octanoyl lysine residue, corresponding in sequence to the lipoyl binding domain of the E2 subunit of pyruvate dehydrogenase. A putative LipA from the hypothermophilic archaea Sulfolobus solfataricus was expressed in Escherichia coli and purified, and the activity was measured using this novel assay. The optimal temperature for the S. solfataricus LipA-dependent formation of the lipoyl group was found to be 60 degrees C.     

N1-aminopropylagmatine, a new polyamine produced as a key intermediate in polyamine biosynthesis of an extreme thermophile, Thermus thermophilus

In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N1-Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N1-Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N1-Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N1-aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N1-aminopropylagmatine to spermidine but does not act on agmatine.     

Metabolic diversification of nitrogen‐containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis

Lysine decarboxylase converts l ‐lysine to cadaverine as a branching point for the biosynthesis of plant Lys‐derived alkaloids. Although cadaverine contributes towards the biosynthesis of Lys‐derived alkaloids, its catabolism, including metabolic intermediates and the enzymes involved, is not known. Here, we generated transgenic Arabidopsis lines by expressing an exogenous lysine/ornithine decarboxylase gene from Lupinus angustifolius (La‐L/ODC) and identified cadaverine‐derived metabolites as the products of the emerged biosynthetic pathway. Through untargeted metabolic profiling, we observed the upregulation of polyamine metabolism, phenylpropanoid biosynthesis and the biosynthesis of several Lys‐derived alkaloids in the transgenic lines. Moreover, we found several cadaverine‐derived metabolites specifically detected in the transgenic lines compared with the non‐transformed control. Among these, three specific metabolites were identified and confirmed as 5‐aminopentanal, 5‐aminopentanoate and δ‐valerolactam. Cadaverine catabolism in a representative transgenic line (DC29) was traced by feeding stable isotope‐labeled [α‐¹⁵N]‐ or [ε‐¹⁵N]‐l ‐lysine. Our results show similar ¹⁵N incorporation ratios from both isotopomers for the specific metabolite features identified, indicating that these metabolites were synthesized via the symmetric structure of cadaverine. We propose biosynthetic pathways for the metabolites on the basis of metabolite chemistry and enzymes known or identified through catalyzing specific biochemical reactions in this study. Our study shows that this pool of enzymes with promiscuous activities is the driving force for metabolite diversification in plants. Thus, this study not only provides valuable information for understanding the catabolic mechanism of cadaverine but also demonstrates that cadaverine accumulation is one of the factors to expand plant chemodiversity, which may lead to the emergence of Lys‐derived alkaloid biosynthesis.