Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph

Summary Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA ^–auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35854, predicted from the deduced amino acid sequence, was similar to the 38 000 M_r determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.     

Spinach leaf dihydrodipicolinate synthase: Partial purification and characterization

The first enzyme unique to lysine biosynthesis in higher plants, dihydrodipicolinate synthase, has been partially purified from spinach leaves, using ion exchange chromatography, hydrophobic interaction chromatography and gel filtration. The spinach enzyme is moderately stable to short-term exposure to heat, in contrast to the pea leaf enzyme, but is unstable on storage even at ?20°. Thiol reagents interfere with the calorimetric assay used, and so cannot be routinely used to stabilize the enzyme, which has an active sulphydryl group. The MW of the enzyme is 115000 (gel filtration). Lysine is a potent inhibitor with an I_(0.5) of 2OμM, whilst the lysine analogue S-β-aminoethylcysteinc has an I_(0.5) of 400 μM. The Kt´_m for aspartic-β-semialdehyde was determined to be 1.4mM, but this compound demonstrated marked substrate inhibition at concentrations above 7 mM, increasing the apparent S_(0.5)for the second substrate, pyruvate.     

Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis

Dihydrodipicolinate synthase (DHDPS) catalyses the first reaction of the (S)-lysine biosynthesis pathway in bacteria and plants. The hypothetical gene for dihydrodipicolinate synthase (dapA) of Thermoanaerobacter tengcongensis was found in a cluster containing several genes of the diaminopimelate lysine–synthesis pathway. The dapA gene was cloned in Escherichia coli, DHDPS was subsequently produced and purified to homogeneity. The T. tengcongensis DHDPS was found to be thermostable (T _0.5 = 3 h at 90°C). The specific condensation of pyruvate and (S)-aspartate-β -semialdehyde was catalyzed optimally at 80°C at pH 8.0. Enzyme kinetics were determined at 60°C, as close as possible to in vivo conditions. The established kinetic parameters were in the same range as for example E. coli dihydrodipicolinate synthase. The specific activity of the T. tengcongensis DHDPS was relatively high even at 30°C. Like most dihydrodipicolinate synthases known at present, the DHDPS of T. tengcongensis seems to be a tetramer. A structural model reveals that the active site is well conserved. The binding site of the allosteric inhibitor lysine appears not to be conserved, which agrees with the fact that the DHDPS of T. tengcongensis is not inhibited by lysine under physiological conditions.     

Catalytic mechanism and cofactor preference of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

Given the rapid rise in antibiotic resistance, including methicillin resistance in Staphylococcus aureus (MRSA), there is an urgent need to characterize novel drug targets. Enzymes of the lysine biosynthesis pathway in bacteria are examples of such targets, including dihydrodipicolinate reductase (DHDPR, E.C. 1.3.1.26), which is the product of an essential bacterial gene. DHDPR catalyzes the NAD(P)H-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) in the lysine biosynthesis pathway. We show that MRSA–DHDPR exhibits a unique nucleotide specificity utilizing NADPH (K_m = 12 μM) as a cofactor more effectively than NADH (K_m = 26 μM). However, the enzyme is inhibited by high concentrations of DHDP when using NADPH as a cofactor, but not with NADH. Isothermal titration calorimetry (ITC) studies reveal that MRSA–DHDPR has ∼20-fold greater binding affinity for NADPH (K_d = 1.5 μM) relative to NADH (K_d = 29 μM). Kinetic investigations in tandem with ITC studies show that the enzyme follows a compulsory-order ternary complex mechanism; with inhibition by DHDP through the formation of a nonproductive ternary complex with NADP⁺. This work describes, for the first time, the catalytic mechanism and cofactor preference of MRSA–DHDPR, and provides insight into rational approaches to inhibiting this valid antimicrobial target.     

Disruption of quaternary structure in Escherichia coli dihydrodipicolinate synthase (DHDPS) generates a functional monomer that is no longer inhibited by lysine

Escherichia coli dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a natively homotetrameric enzyme was converted to a monomeric species through the introduction of destabilising interactions at two different subunit interfaces allowing exploration of the roles of the quaternary structure in affecting catalytic competency. The double mutant DHDPS-L197D/Y107W displays gel filtration characteristics consistent with a single non-interacting monomeric species, which was confirmed by sedimentary velocity experiments. This monomer was shown to be catalytically active, but with reduced catalytic efficiency (k_cat = 9.8 ± 0.5 s⁻¹), displaying 8% of the specific activity of the wild-type enzyme. The Michaelis constants for the substrates pyruvate and for (S)-aspartate semialdehyde increased by an order of magnitude, indicating that quaternary structure plays a significant role in substrate specificity. This monomeric species exhibited an enhanced propensity for aggregation and inactivation, indicating that whilst the oligomerization is not an intrinsic criterion for catalysis, higher oligomeric forms may benefit from both increased catalytic efficiency and diminished aggregation propensity. Furthermore, allosteric inhibition by (S)-lysine was abolished for DHDPS-L197D/Y107W, confirming the importance of the dimeric unit as the minimal functional assembly for efficient (S)-lysine binding.     

Purification and characterization of nitric oxide synthase from Staphylococcus aureus

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.     

Lysine overproducer mutants with an altered dihydrodipicolinate synthase from protoplast culture of Nicotiana sylvestris (Spegazzini and Comes)

Summary Two S-(2-aminoethyl)L-cysteine (AEC) resistant lines were isolated by screening mutagenized protoplasts from diploid N. sylvestris plants. Both lines accumulated free lysine at levels 10 to 20-fold higher than in controls. Lysine overproduction and AEC-resistance were also expressed in plants regenerated from the variant cultures. A feedback insensitive form of dihydrodipicolinate synthase (DHPS), the pathway specific control enzyme for lysine synthesis, was detected in callus cultures and leaf extracts from the resistant lines. Aspartate kinase (AK), the other key enzyme in the regulation of lysine biosynthesis, was unaltered in the mutants. Crosses with wild type plants indicated that the mutation conferring insensitivity to feedback in DHPS, with as result overproduction of lysine and resistance to AEC, was inherited as a single dominant nuclear gene.Abbreviations AK aspartate kinase (EC 2.7.2.4) - DHPS dihydrodipicolinate synthase (EC 4.2.1.52) - AEC S-(2-aminoethyl)L-cysteine     

Lysine accumulation in maize cell cultures transformed with a lysine-insensitive form of maize dihydrodipicolinate synthase

Lysine is one of the nutritionally limiting amino acids in food and feed products made from maize (Zea mays L.). Two enzymes in the lysine biosynthesis pathway, aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS), have primary roles in regulating the level of lysine accumulation in plant cells because both enzymes are feedback-inhibited by lysine. An isolated cDNA clone for maize DHPS was modified to encode a DHPS much less sensitive to lysine inhibition. The altered DHPS cDNA was transformed into maize cell suspension cultures to determine the effect on DHPS activity and lysine accumulation. Partially purified DHPS (wildtype plus mutant) from transformed cultures was less sensitive to lysine inhibition than wild-type DHPS from nontransformed cultures. Transformed cultures had cellular free lysine levels as much as four times higher than those of nontransformed controls. Thus, we have shown that reducing the feedback inhibition of DHPS by lysine can lead to increased lysine accumulation in maize cells. Increasing the capacity for lysine synthesis may be an important step in improving the nutritional quality of food and feed products made from maize.     

Conserved main-chain peptide distortions: a proposed role for Ile203 in catalysis by dihydrodipicolinate synthase

In recent years, dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) has received considerable attention from a mechanistic and structural viewpoint. DHDPS catalyzes the reaction of (S)-aspartate-beta-semialdehyde with pyruvate, which is bound via a Schiff base to a conserved active-site lysine (Lys161 in the enzyme from Escherichia coli). To probe the mechanism of DHDPS, we have studied the inhibition of E. coli DHDPS by the substrate analog, beta-hydroxypyruvate. The K (i) was determined to be 0.21 (+/-0.02) mM, similar to that of the allosteric inhibitor, (S)-lysine, and beta-hydroxypyruvate was observed to cause time-dependent inhibition. The inhibitory reaction with beta-hydroxypyruvate could be qualitatively followed by mass spectrometry, which showed initial noncovalent adduct formation, followed by the slow formation of the covalent adduct. It is unclear whether beta-hydroxypyruvate plays a role in regulating the biosynthesis of meso-diaminopimelate and (S)-lysine in E. coli, although we note that it is present in vivo. The crystal structure of DHDPS complexed with beta-hydroxypyruvate was solved. The active site clearly showed the presence of the inhibitor covalently bound to the Lys161. Interestingly, the hydroxyl group of beta-hydroxypyruvate was hydrogen-bonded to the main-chain carbonyl of Ile203. This provides insight into the possible catalytic role played by this peptide unit, which has a highly strained torsion angle (omega approximately 201 degrees ). A survey of the known DHDPS structures from other organisms shows this distortion to be a highly conserved feature of the DHDPS active site, and we propose that this peptide unit plays a critical role in catalysis.     

Purification and characterization of dihydrodipicolinate synthase from pea

Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis in bacteria and higher plants, has been purified to homogeneity from etiolated pea (Pisum sativum) seedlings using a combination of conventional and affinity chromatographic steps. This is the first report on a homogeneous preparation of native dihydrodipicolinate synthase from a plant source. The pea dihydrodipicolinate synthase has an apparent molecular weight of 127,000 and is composed of three identical subunits of 43,000 as determined by gel filtration and cross-linking experiments. The trimeric quaternary structure resembles the trimeric structure of other aldolases, such as 2-keto-3-deoxy-6-phosphogluconic acid aldolase, which catalyze similar aldol condensations. The amino acid compositions of dihydrodipicolinate synthase from pea and Escherichia coli are similar, the most significant difference concerns the methionine content: dihydrodipicolinate synthase from pea contains 22 moles of methionine residue per mole of native protein, contrary to the E. coli enzyme, which does not contain this amino acid at all. Dihydrodipicolinate synthase from pea is highly specific for the substrates pyruvate and l-aspartate-β-semialdehyde; it follows Michaelis-Menten kinetics for both substrates. The pyruvate and l-aspartate-β-semialdehyde have Michaelis constant values of 1.70 and 0.40 millimolar, respectively. l-Lysine, S-(2-aminoethyl)-l-cysteine, and l-α-(2-aminoethoxyvinyl)glycine are strong allosteric inhibitors of the enzyme with 50% inhibitory values of 20, 160, and 155 millimolar, respectively. The inhibition by l-lysine and l-α-(2-aminoethoxyvinyl)glycine is noncompetitive towards l-aspartate-β-semialdehyde, whereas S-(2-aminoethyl)-l-cysteine inhibits dihydrodipicolinate synthase competitively with respect to l-aspartate-β-semialdehyde. Furthermore, the addition of (2R,3S,6S)-2,6-diamino-3-hydroxy-heptandioic acid (1.2 millimolar) and (2S,6R/S)-2,6-diamino-6-phosphono-hexanic acid (1.2 millimolar) activates dihydrodipicolinate synthase from pea by a factor of 1.4 and 1.2, respectively. This is the first reported activation process found for dihydrodipicolinate synthase.     

Isolation and characterization of dihydrodipicolinate synthase from maize

Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme specific to lysine biosynthesis in plants, was purified from maize (Zea mays L.) cell suspension cultures and leaves. The subunit molecular weight of maize dihydrodipicolinate synthase was estimated to be 38,000 based on SDS-PAGE. The condensation of l-aspartate semialdehyde and pyruvate by highly purified dihydrodipicolinate synthase exhibited kinetics characteristic of a Ping Pong Bi Bi ordered reaction in which pyruvate binds first to the enzyme. Substrate inhibition evident at higher concentrations of l-aspartate semialdehyde was partially alleviated by increasing concentrations of pyruvate. Pyruvate binding exhibited cooperativity with an apparent number of 2 and 1.86 millimolar concentration required for 50% of maximal activity. The K_m for aspartate semialdehyde was estimated to be 0.6 millimolar concentration. Lysine was an allosteric cooperative inhibitor of dihydrodipicolinate synthase with an estimated Hill number of 4 and 23 micromolar concentration required for 50% inhibition. The physical and kinetic data are consistent with a homotetramer model for the native enzyme.     

The crystal structure of three site-directed mutants of Escherichia coli dihydrodipicolinate synthase: further evidence for a catalytic triad

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes: the condensation of (S)-aspartate-beta-semialdehyde and pyruvate. The crystal structure of wild-type DHDPS has been published to 2.5A, revealing a tetrameric molecule comprised of four identical (beta/alpha)(8)-barrels, each containing one active site. Previous workers have hypothesised that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Tyr133, Thr44 and Tyr107, which provide a proton shuttle to transport protons from the active site to solvent. We have tested this hypothesis using site-directed mutagenesis to produce three mutant enzymes: DHDPS-Y133F, DHDPS-T44V and DHDPS-Y107F. Each of these mutants has substantially reduced activity, consistent with the catalytic triad hypothesis. We have determined each mutant crystal structure to at least 2.35A resolution and compared the structures to the wild-type enzyme. All mutant enzymes crystallised in the same space group as the wild-type form and only minor differences in structure are observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS.     

Geranyl diphosphate synthase from Abies grandis: cDNA isolation,functional expression,and characterization

Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library. When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1). Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.     

Cloning of Lactobacillus plantarum IAM 12477 lysine biosynthetic genes encoding functional aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, and dihydrodipicolinate reductase

Summary The lysine biosynthetic genes asd, dapA, and dapB, encoding aspartate semialdehyde dehydrogenase (ASADH), dihydrodipicolinate synthase (DHPS), and dihydrodipicolinate reductase (DHPR), respectively, have been cloned from Lactobacillus plantarum IAM 12477 by heterologous complementation to Escherichia coli mutants. The amino acid sequences of the cloned genes showed considerable similarities to the corresponding protein from other gram-positive bacteria. We identified the amino acids that correspond to key catalytic residues of ASADH, DHPS, and DHPR and found them to be conserved in the protein from L. plantarum. ASADH, DHPS, and DHPR activity was detected in the cell extracts of E. coli mutant harboring each gene, indicating that the cloned genes were functionally expressed in E. coli. The regulation of ASADH, DHPS, and DHPR were studied in the cell extracts of both the E.␣coli mutant harboring the gene and L. plantarum; however, those enzymes were found not to be regulated by the end products of the pathway. This paper represents a portion of the thesis submitted by M. N. Cahyanto to Osaka University as partial fulfillment of the requirements for the PhD degree.     

Structural characterization of the alpha-hemolysin monomer from Staphylococcus aureus

Alpha-hemolysin from Staphylococcus aureus is secreted as a water-soluble monomer and assembles on membranes to oligomerize into a homo-heptameric, water-filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X-ray crystallography, structures of intermediate states-from the soluble monomer to all potential "pre-pore" structures-are yet unknown. Here, we propose a model of the monomeric alpha-hemolysin in solution based on molecular modeling, verified by small angle X-ray scattering data. This structure reveals details of the monomeric conformation of the alpha-hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates.     

Purification and characterization of dihydrodipicolinate synthase from wheat suspension cultures

Dihydrodipicolinate synthase, the first enzyme unique to lysine biosynthesis in higher plants, was purified about 5100-fold from suspension-cultured cells of wheat (Triticum aestivum var Chinese Spring). The synthase has an average molecular weight of 123,000 as determined by gel filtration and exhibited maximum activity at pH 8.0. The kinetics of the condensation reaction are compatible with a “Ping Pong” mechanism in which pyruvate reacts first with the enzyme to form a Schiff base. Pyruvate and l-aspartic-β-semialdehyde (ASA) have respective K_m values of 11.76 and 0.80 millimolar. Allosteric inhibition was observed with increasing concentrations of l-lysine and its structural analogs, including threo-4-hydroxy-l-lysine and S-(2-aminoethyl)-l-cysteine, with respective I_0.5 values of 51, 141, and 288 micromolar. These amino acids were competitive inhibitors with respect to ASA and noncompetitive inhibitors with respect to pyruvate. We propose that the binding site for lysine overlaps with the ASA binding site, possibly by an attachment of the common alanyl moiety. The wheat enzyme was inhibited by Zn²⁺, Cd²⁺, and Hg²⁺ and also by sulfhydryl inhibitors, p-(hydroxymercuri)benzoic acid and p-chloromercuribenzenesulfonic acid.     

Dihydrodipicolinate synthase ofnicotiana sylvestris,a chloroplast-localized enzyme of the lysine pathway

The first enzyme of the lysine-biosynthesis pathway, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has been purified and characterized inNicotiana sylvestris Speggazini et Comes. A purification scheme was developed for the native DHDPS that subsequently led to the purification to homogeneity of its subunits using two-dimensional gel electrophoresis. Subsequent elution of the purified polypeptide has opened the way for the production of rabbit polyclonal anti-DHDPS sera. The molecular weight of the enzyme was determined to be 164000 daltons (Da) by an electrophoretic method. By labeling with [¹⁴C]pyruvate, the enzyme was shown to be composed of four identical subunits of 38500 Da. Pyruvate acts as a stabilizing agent and contributes to the preservation of the tetrameric structure of the enzyme. The enzyme ofN. sylvestris is strongly inhibited by lysine with anI _0.5 of 15 μM; S-(2-aminoethyl)L-cysteine and γ-hydroxylysine, two lysine analogs, were found to be only weak inhibitors. An analog of pyruvate, 2-oxobutyrate, competitively inhibited the enzyme and was found to act at the level of the pyruvate-binding site. Dihydrodipicolinate synthase was localized in the chloroplast and identified as a soluble stromal enzyme by enzymatic and immunological methods. Its properties are compared with those known for other plant and bacterial DHDPS enzymes.